Patent Application
20230101856
This invention concerns a versatile and simple one-pot method to prepare nanomaterials, and in particular nanoparticles, grafted with an ultra-thin layer of calixarenes as well as a new process for manufacturing these materials. The invention further concerns the use of these calixarene-grafted nanomaterials.
The use of nanomaterials, and in particular metal-based nanomaterials has become widespread in many technical fields, from biomedical applications or paints to microelectronics or as chemical catalysts. Their preparation is however not always straightforward, due to stability issues or difficulty to control their size and/or shape. Their functionalization is not simpler for the same stability reasons, and because reactive groups have to be introduced at the surface of the nanomaterial.
For example, silver nanoparticles are known to have anti-bacterial effect, which in part results from their instability and release of silver ions. This same instability prevents them from being functionalized. It is therefore so far extremely difficult to prepare functionalized silver nanoparticles.
One class of organic substances that has been proposed for immobilizing or grafting onto material surfaces is that of the calix[n]arenes (or calixarenes). Calix[n]arenes are cyclic phenoxy derivatives in which n is the number of phenoxy groups, linked in their ortho positions by methylene bridges or sulfur bridges in the case of thiacalixarenes. Calixarenes are conformationally flexible molecules that can display different conformations due to the ability to undergo complete ring inversions. Calixarenes can eventually possess a cup-like structure (i.e. cone conformation) having a narrow and a large rim.
In the following, the terms “calix[n]arenes” and “calixarenes” refer to both families of compounds, those with methylene bridges (named calix[n]arenes), and those with S bridges (named thiacalix[n]arenes) and their oxide derivatives (SO and SO2 bridges). In addition, calix[n]arenes also have varying numbers of phenoxy moieties expressed by the symbol [n], wherein n represents the number of phenoxy moieties, in particular n can be 4, 5, or 6. Calix[n]arenes are known compounds that have been synthesized with various substitution patterns, for example with substituents on the aromatic part of the phenoxy moieties or on their hydroxyl groups. These cyclic compounds can find various applications in a manifold of areas, including the development of enzyme mimetics, ion sensitive electrodes or sensors, selective membranes, non-linear optics, and HPLC stationary phases.
Calix[n]arenes have been used as coatings on various materials. The immobilization of (thia)calixarenes onto a surface has been reported using different attachment techniques. The resulting immobilized calixarenes were used as receptors.
EP2836539 discloses a grafting method to coat an ultrathin layer of calixarenes on the surface of a material. The calixarenes are grafted via the large rim, using diazonium chemistry, and it leads to rigid and stable molecular layers, which offer a robust and stable platform for further (post-)functionalization. The macrocyclic structure of the calixarene prevents polymerization during the grafting process, induces spatial pre-organization and pre-structuration and allows the orthogonal polyfunctionalization of the platforms, with a precise spatial control.
Though leading to robust grafting, this method can only be efficient for stable nanomaterials, and could, for example not be applied to silver nanostructures.
There is a need for a more versatile and tunable method to prepare nanomaterials functionalized with calixarenes, with a highly robust, structurally regular, ultra-thin layer, in a controlled manner. Indeed, for certain applications, it is important to control the size and shape of the nanomaterial as well as the surface density, e.g. for applications as sensors, because this may have an impact on the detection sensitivity and efficiency.
In accordance with the present invention, a one-pot synthesis gives nanomaterials grafted or coated with an ultra-thin layer of calix[n]arenes from a metallic salt (or a mixture of metallic salts) and a calixarene-diazonium salt (or a mixture of calixarene-diazonium salts), in reductive conditions. This leads to the formation of a robust nanomaterial coated with an ultra-thin layer, which may be rather dense and which, if desired, can serve as a platform for one or more further functionalization(s).
To this purpose, the invention relates to a method to synthesize metal-based nanomaterials coated with calix[n]arenes comprising the steps of:
Using this method, the calix[n]arenes are bound onto the metallic surface of the nanomaterial with covalent bonds.
The method of the invention can further comprise the step of adjusting the value of the pH of the reaction mixture preferably between 3.5 and 10, preferably between 4.5 and 9.5, preferably between 5.5 and 9, preferably around 8,
The molar ratio of metal-based salt to calixarenes influences the number of particles formed and hence their size, as well as the quality of the coating. A too large excess of calixarene diazonium may lead to the calixarene reacting with itself. This ratio should preferably be fixed between 5:1 and 1:5, preferably between 4:1 and 1:4, preferably between 2:1 and 1:2, still preferably around 1:1.
The reducing agent can be any reducing agent found suitable by the person skilled in the art like for example hydrides (e.g. sodium borohydride, sodium cyanoborohydride, . . . ), ascorbate salts (e.g. sodium ascorbate), etc. A combination of reducing agents can also be used, in particular, for example a combination comprising ascorbate. The strength and the amount of reducing agent can be modified in order obtain different sizes: weaker reducing agents or lower amounts of it will lead to bigger nanoparticles. The molar proportion of reducing agent to calixarene-diazonium salt is preferably comprised between 20:1 and 1:10, preferably between 15:1 and 1:5, preferably between 10:1 and 1:4, preferably between 5:1 and 1:2, still preferably around 4:1. The person skilled in the art will know how to adapt proportions, taking for example into account the number of hydrides a given reducing agent can provide.
Heating is preferably performed under mixing, at temperatures comprised between 15° C. and 150° C., preferably between 20° C. and 120° C., preferably between 25° C. and 100° C., preferably between 40 and 80° C. and still preferably around 60° C. It was observed by the applicants that the reaction temperature has an impact on the particle size distribution.
A reaction temperature of around 60° C. leads to a narrow size distribution.
Reaction time is at least 1 h, preferably at least 2 h or at least 3 h, still preferably at least 5 h or at least 8 h and up to 48 h, up to 24 h, preferably up to 16 h. The applicant has noted that after 16 h, the formation of the nanoparticles was completed as well as the grafting of the calixarenes onto these particles.
The invention also relates to the metal-based nanomaterials coated with calix[n]arenes obtained by the process of the invention, which have the distinctive features over nanomaterials obtained by other methods to have an homogeneous particles distribution (i.e. above 80% of the particle formed have the same average dimension ±10%). Moreover, the metal-based nanomaterials of the invention are coated only with calix[n]arenes, meaning that the only ligands at the surface of the metal are calixarenes. Indeed, with other methods, where a ligand like citrate, present at the surface of a particle, is interchanged with a calixarene, there are always some residual citrate ligands at the surface of the metal. The metal-based nanomaterials of the invention can therefore be distinguished from metal-based nanomaterials prepared by ligand-exchange techniques by the absence of residual ligands other than calixarenes at their surface.
Preferably, the oxidized metal MX is a metal oxide or a mixture of metal oxides, or a salt of a metal or a mixture of salts.
The salt generally implies that the metallic part is positively charged, the counter ion can be any suitable ion. The metal-based salt can for example be a halide, a nitrate, a sulfate, a carboxylate, a triflate, an alcoolate, an hydroxylate, a sulfonate, a phosphate (like hexafluorophosphate), a tosylate, a borate (like tetrafluoroborate) . . . . In some cases, the salt can be not clearly ionized, such as for example silicate organic salts like tetraethyl orthosilicate (TEOS), trimethyl orthosilicate (TMOS), . . . .
An halide is preferably a chloride, a fluoride, a bromide or an iodide.
The oxidized metal is not necessarily soluble in water. For example, hexafluorophosphate salts or tetrafluoroborate salts can be suitable in organic solvents.
A carboxylate can be any suitable carboxylate ion, derived from a carboxylic acid, preferably a C1-C20, still preferably a C1-C10, branched or unbranched, substituted or unsubstituted, carboxylic acid and more preferably, the carboxylate is formate, acetate, propionate, butyrate, lactate, oxalate, citrate, trifluoroacetate or oxalate.
A metal can be any of the metals as defined in the periodic table of the elements, an in particular of the subclasses alkali metals, alkaline earth metals, lanthanides, actinides, transition metals, post transition metals, or metalloids. Preferably, the metal is a transition metal or a post transition metal. Preferably, the metal is selected from the list comprising silver, palladium, gold, platinum, copper, nickel, zinc, cadmium, indium, lead, aluminum, titanium, silicon, tantalum or iron.
A metal oxide is any oxide of the metals as defined above. Preferably, a metal oxide is a transition metal oxide, such as, but not limited to, tisane oxide, tantalum oxide, iron oxide, copper oxide, silver oxide, nickel oxide or a post transition metal oxide, such as, but not limited to, silicon oxide, zinc oxide, cadmium oxide, indium oxide, lead oxide or aluminum oxide.
An alloy or mixture of metals is a combination of two or more metals and/or metal oxides as defined above. In some cases, an alloy comprises at least two metals and/or metal oxides from the same subclasses or from different subclasses. Preferably, an alloy comprises at least two metals or metal oxides from the subclass transition metal and/or post transition metal. Preferably, the alloy comprises at least two metals and/or metal oxides selected from the list comprising silver, palladium, gold, platinum, copper, nickel, zinc, cadmium, indium, lead, aluminum, iron, titanium, silicon, tantalum, and their respective oxides. A metal base salt is for example silver nitrate, palladium dichloride, platinum dichloride, chloroauric acid, copper (II) acetate, iron (II) chloride, iron (III) chloride.
A nanomaterial according to the present invention is a particulate material (nanoparticle) having at least one dimension in the nanometric range, i.e. between 1 and 999 nm, preferably between 5 and 800 nm, still preferably between 10 and 500 nm, preferably below 250 nm or below 150 nm or below 100 nm as generally accepted for a nanomaterial. A nanomaterial according to the present invention can have any shape, like for example, but not limited to a spherical shape, a cubical shape, a star shape, a rod shape, a wire shape, a nanocage or a triangular shape. The final shape of the nanomaterial is dependent on the nature of the metal-based element(s), the reacting conditions like pH, nature of the reducing agent, temperature, addition rate of the reducing agent, . . . .
The metal-based nanometarial core can be designed to have various properties by including one or more elements conferring for example optical properties (with e.g. gold, silver, copper, platinum), magnetic properties (with e.g. iron oxide), catalytic properties (with e.g. copper, palladium, titanium, platinum, tantalum) or antimicrobial properties (with e.g. silver or copper).
Calix[n]arenes are organic macrocycles wherein four or more phenolic structures are linked so as to form a crown, the linker between the phenolic structures usually consisting of CH2, S, SO or SO2. n designs the number of phenolic structures comprised in the macrocycle. n is typically comprised between 4 and 6.
When present on a nanomaterial surface, each of the aromatic subunits of the calixarene can adopt either an “up” or a “down” orientation towards the grafted surface. “Up” orientation refers to the phenolic groups pointing in the direction of the surface and “down” orientation refers to the phenolic groups pointing in the opposite direction, away from the surface.
A calix[n]arene diazonium salt designs a calix[n]arene wherein at least one phenolic ring is functionalized with a diazonium salt, as represented in formula I.
In one embodiment:
X represents CH2, S, SO or SO2; when X is S, the calixarene can be called a thiacalixarene;
R1, R2, R3, R4, R5 and R6 each independently represent hydrogen or C1-30 alkyl optionally substituted with one or more substituents each independently selected from the group consisting of halo (e.g. fluoro, chloro, bromo, iodo), carboxylic acid ester, alkyl or benzyl thioester, alkenyl, alkynyl, C1-30 alkoxy, aryl, substituted aryl (wherein the substituent is fluoro or cyano or C1-30 alkyl or C1-30 alkoxy), —N3, cyano, carboxylic acid, carboxylic acid amide, —OH, amino, amido, imino, carbamate, acyl chloride, ureido, thioureido, mercapto, substituted disulfide, maleimide, heterocyclic, amino acid and amino acid derivative, peptide, protein, DNA, RNA, microRNA, phosphine or phosphine oxide, crown ether, aza-crown ether, cryptand, porphyrin, calixarene, cyclodextrin, resorcinarene, saccharide, and polyethylene glycol; and wherein two or more of R1, R2, R3, R4, R5 and R6 may be covalently linked either directly or by a bridge that includes oxygen, phosphine, phosphine oxide, sulfur, SO, SO2, amino, imino, amido, ureido, thioureido, ester, thioester, alkene, alkyne or alkyl;
Y2—, Y3—, Y4— Y5— and Y6— are each independently selected from the group consisting of hydrogen, diazonium salt, OH, NO2, halogen, C1-30 alkyl, acyl, carboxylic acid and derivatives (e.g. ester, amide), —N3 alkenyl or alkynyl;
Y1— is a diazonium salt N2+X− where X− represents an anion such as but not limited to BF4−, PF6−, Cl−, TsO− (tosylate).
The index “0,1” at the right side of the aryl moieties bearing R5 and R6 in formula I means “0 or 1”, meaning that these aryl moieties, each independently, can be present or absent.
The synthesis of calix[n]arene-diazonium salts can be performed either by in situ diazotation of the amino groups or by diazotation followed by isolation of the diazonium salts of formula I, according to procedures known in the art, as for example disclosed in EP2836539.
The calix[n]arenes bound to the surface of the metal-based nanomaterial is at least one of the compounds of formula (II) wherein:
X represents CH2, S, SO or SO2;
R1, R2, R3, R4, R5 and R6 each independently represent hydrogen or C1-30 alkyl optionally substituted with one or more substituents each independently selected from the group consisting of halo (e.g. fluoro, chloro, bromo, iodo), carboxylic acid ester, alkyl or benzyl thioester, alkenyl, alkynyl, C1-30 alkoxy, aryl, substituted aryl (wherein the substituent is fluoro or cyano or C1-30 alkyl or C C1-30 alkoxy), —N3, cyano, carboxylic acid, carboxylic acid amide, —OH, amino, amido, imino, carbamate, acyl chloride, ureido, thioureido, mercapto, substituted disulfide, maleimide, heterocyclic, amino acid and amino acid derivative, peptide, protein, DNA, RNA, microRNA, phosphine or phosphine oxide, crown ether, aza-crown ether, cryptand, porphyrin, calixarene, cyclodextrin, resorcinarene, saccharide, and polyethylene glycol; and wherein two or more of R1, R2, R3, R4, R5 and R6 may be covalently linked either directly or by a bridge that includes oxygen, phosphine, phosphine oxide, sulfur, SO, SO2, amino, imino, amido, ureido, thioureido, ester, thioester,alkene, alkyne or alkyl;
Z2—, Z3—, Z4—, Z5— and Z6— are each independently selected from the group consisting of a covalent bond as defined for Z1, hydrogen, diazonium salt, OH, NO2, halogen, C1-30 alkyl, acyl, carboxylic acid and derivatives (e.g. ester, amide), —N3 alkenyl or alkynyl;
Z1— is a covalent bond with the metal of the nanomaterial surface, said bond being a direct or indirect covalent bond.
A direct covalent bond, between the aromatic ring and the metal of the nanomaterial core may be obtained by loss of N2 during the synthesis process. An indirect covalent bond may be obtained when N2 is not lost during the synthesis process and remains as a linker between the aromatic ring and the metal.
The formation of a direct or indirect covalent bond may depend on the nature of the metal, the nature of the calixarene and/or the synthetic conditions.
It will be appreciated that groups having similar references in formula (I) and formula (II) are indeed similar, the nanomaterial of formula (II) resulting from the reaction of at least one of the diazonium salts of formula (I) according to the method of the invention. The following part of the present description will therefore refer to both compounds of formula (I) and compounds of formula (II). Chemical groups differing between the diazonium salt of formula (I) and the nanomaterial of formula (II) are noted differently.
Also preferably, at least one of Y2—, Y3—, Y4— Y5— and Y6— (in addition to Y1—) is a diazonium salt.
Conversely, at least one of Z2—, Z3—, Z4— Z5— and Z6 is a bond with the metal of the nanomaterial surface.
Possibly, the calix[n]arene of formula I bears one, two, three, four or, if applicable, five or six diazonium groups, and an equal number of bonds are formed with the surface of the material. This may be applicable for the less flexible calix[n]arenes, in particular where n is 4, or with appropriately substituted calix[n]arenes. This may also be applicable for the more flexible calix[n]arenes, such as the calix[5]arenes or the calix[6]arenes which can be modified by adding appropriate substituents on the small rim or covalent bridges between the phenolic moieties (i.e. where two or more of R1, R2, R3, R4, R5 and R6 are covalently linked either directly or through a bridge as defined above).
The skilled person will be able to select the number of bonds to the surface per calixarene moiety based on the conformational flexibility of the (thia)calix[n]arene moiety, or on the possibilities in terms of chemical configuration, in particular as regards steric hindrance.
Preferably, the calix[n]arene is a calix[4]arene.
Preferably, the calix[n]arene is a calix[4]arene wherein at least one of Y2—, Y3— and Y4— is a diazonium salt, preferably at least two of Y2—, Y3— and Y4— are diazonium salts, preferably Y2—, Y3— and Y4— are diazonium salts.
Preferably, R1, R2, R3, R4, R5 and R6 each independently represent a C1-30 alkyl optionally substituted with one or more R substituents each independently selected from the group consisting of C1-30 alkoxy, —N3, cyano, carboxylic acid, carboxylic acid amide, acid halide, amide ester, —OH, amino, amido, imino, carbamate, maleimide, thiol, cyanate, isocyanate or acyl chloride.
Preferably, R1, R2, R3, R4, R5 and R6 each independently represent polysaccharides, CH2COOH, CH2(CH2OCH2)mCH2OCH3, CH2(CH2OCH2)pCH2OCH2COOH or CH2(CH2OCH2)qCH2R, wherein R is as defined above, wherein m, p and q are not limited but are, each independently, preferably 50 or below.
The metal-based nanomaterial MC of the invention is coated with calix[n]arenes, meaning that a particulate nanomaterial is formed with a metallic central core M having calix[n]arenes bound to its surface so as to form an ultrathin layer C, as illustrated in Formula III.
The ultrathin layer of calix[n]arenes is preferably a monolayer, meaning that the thickness of the ultrathin monolayer is the thickness of a calix[n]arene, including its substituents and functional groups.
Combinations of metal-based salts can be used, leading to the formation of mixed metal-based central cores, i.e. metal alloys.
Combinations of calix[n]arenes-diazonium salts can be used, leading to the formation of mixed layers whose composition can be controlled. As calix[n]arenes can be further functionalized, depending on the nature of R1, R2, R3, R4, R5 and R6, this presents the advantage of offering the possibility to confer a plurality of functionalities to the metal-based nanomaterials coated with calix[n]arenes.
The invention also relates the metal-based nanomaterials of the invention for use in immunoassays. The immunoassays are preferably immunoturbidimetry tests or lateral flow immune-assays (LFIAs).
The metal-based nanomaterials of the invention for use in immunoassays are preferably silver based and/or gold-based nanomaterials. Still preferably, the metal-based nanomaterials of the invention for use in immunoassays is a silver-based nanomaterial. So far, silver-based nanomaterials were usually not stable enough to be used in such kind of applications. Thanks to the method of the invention, this problem is overcome, and silver nanoparticles coated with calixarenes can be reliably produced and conjugated with biomolecules. The sensitivity of an immunoassay using silver based calixarene coated nanomaterials has been found to be much higher that the sensitivity of the same using gold.
The metal-based nanomaterials of the invention for use in immunoassays can be used to detect anti-SARS-CoV-2 human IgG (i.e. anti-SARS-CoV-2 human IgG: Anti-Spike-RBD fully human mAb(IgG )) and/or IgM and/or the viral Protein S (i.e. Recombinant SARS-CoV-2, S1 Subunit Protein (RBD)).
The metal-based nanomaterials coated with calix[n]arenes of the invention for use in immunoassays are preferably coated with calix[4]arenes.
The metal-based nanomaterials coated with calix[n]arenes of the invention for use in immunoassays are preferably bio-conjugated with a biomolecule, like a protein, the protein being bound to at least one of R1, R2, R3, R4, R5 and R6. The protein is preferably an immunoglobulin or any protein to be detected.
Bioconjugation can be done using various classical techniques, including, but not limited to, passive adsorption, peptide coupling, copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), or others (maleimide-thiol, hydrazine-aldehyde, . . . ) . . . .
The term “about” when used in relation to a numerical value has the meaning generally known in the relevant art. In certain embodiments the term “about” may be left out or it may be interpreted to mean the numerical value+10%; or +5%; or +2%; or +1%.
The term “thickness” refers to the distance between the surface that is grafted and the part of the grafted (thia)calix[n]arene molecule that is furthest away from the surface. Usually an ultrathin layer of calixarene is grafted onto the metallic core. The ultrathin layer is preferably a monolayer. An ultrathin layer has a thickness of about 1 nm to 15 nm, corresponding to the calixarene rings without any further substituents (i.e. not counting R1, R2, R3, R4, R5 and R6 groups)
The terms “rather dense” and “dense” are used to describe a surface of a material that is coated with molecules in such way that molecules considered as single spheres or cylinders occupy an area equivalent to more than 50%, or more than 60%, or more than 70%, of a close-packed organization of the spheres or cylinders according to the compact van der Waals model.
The term “alkyl” refers to non-aromatic hydrocarbon groups. In particular “alkyl” refers to linear or branched, cyclic (e.g. cycloalkyl) and non-cyclic (acyclic) hydrocarbon groups. These may be unsaturated (see “alkenyl” and “alkynyl” below) or saturated. They can have varying numbers of carbon atoms, e.g. up to about 30, or up to about 20, or up to about 15, or up to about 10 carbon atoms. Alkyl groups thus include C1-30 alkyl, C1-10 alkyl (as more specifically defined below), C1-6 alkyl, and C1-4 alkyl groups.
The term “C1-10 alkyl” denotes straight and branched saturated hydrocarbon radicals having from one to ten carbon atoms such as, for example, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-butyl, 2-methylpropyl, 1-pentyl, 2-pentyl, 2-methylpropyl, 1-hexyl and other hexyl isomers, 1-heptyl and other heptyl isomers, 1-octyl and other octyl isomers, 1-nonyl and other nonyl isomers, 1-decyl and other decyl isomers. The term “C1-6 alkyl” include one to six carbon atoms. “C1-4alkyl” have from one to four carbon atoms. Of particular interest are straight (non-branched) C1-10 alkyl, C1-6 alkyl, or C1-4 alkyl groups.
In the embodiments where two or more of R1, R2, R3, R4, R5 and R6 form a bridge (bridging group) selected from phosphine, phosphine oxide, amino, imino, amido, ureido, thioureido, ester, thioester, alkene, alkyne or alkyl, one of the hydrogen atoms or substituents on these moieties are replaced by a bond.
The calix[n]arene-based diazonium salts of formula I can be prepared by reacting a calix[n]arene bearing amino NH2 groups with a nitrite such as sodium nitrite, in an aqueous acidic solution or with an alkyl nitrite such as isoamylnitrite or tertio-butylnitrite in an organic solvent (e.g. dichloromethane, polar aprotic solvents such as acetonitrile, dimethylformamide, dimethylacetamide, and the like solvents) or with nitroso salt (NOX) in organic solvents (e.g. acetonitrile). The diazonium salts of formula I, preferably the BF4− salts, can be prepared from an ice-cold solution of the corresponding amino NH2 derivatives in HBF4 by the slow addition of NaNO2 (in excess) dissolved in a minimum amount of water. The precipitate is filtered off, washed with H2O. The diazonium salts of formula I, preferably its BF4− salts, can be prepared from a solution of the corresponding amino NH2 derivatives in acetonitrile in the presence of NOBF4 (preferably in a slight molar excess) at low temperature (e.g. −40° C.). Typically, the crude residue is then washed with diethylether and/or ethanol.
The ultrathin layer of grafted (thia)calix[n]arenes is a homogeneous layer and does not present the large ramifications typically encountered with other systems, which lead to a complex and irregular surface coating. The grafted (thia)calix[n]arenes may form a rather dense coating, so that little free surface of the coated material is present.
Classical (bio)conjugation reactions such as peptide type coupling, maleimide-thiol reaction or copper catalyzed Huisgen cycloaddition (click chemistry) can be used for the immobilization of molecules or biomolecules on the grafted (thia)calix[n]arene ultrathin layer, the choice of the reaction depending on the groups present on the grafted (thia)calix[n]arenes. As a representative example, the grafted (thia)calix[n]arene ultrathin layer, when functionalized with a carboxylic acid group, can be esterified or converted with an appropriate amine into amides. Appropriate amines include not only simple amines but also amino acids, peptides, proteins, immunoglobulins, DNA, RNA, microRNA, and various chemical species (such as ligands for metal ions or for anions, molecular receptors, oligomers or polymers) with one or multiple appending amino arms. The carboxylic acid group can further be linked to hydroxyl-containing species such as saccharides, cyclodextrins and polyethylene glycols.
The covalent surface grafting of functionalized (thia)calix[n]arenes (with e.g. COOH, maleimide or alkyne groups on the small rim) on the large rim provides well-organized and compact monolayers, which can be post-functionalized. In other words, grafted (thia)calix[n]arenes induce a pre-structuration and a pre-functionalization of the surface at the molecular level.
The grafted materials of the invention can be used as a versatile platform for further modification, in particular the anchoring of further molecules resulting a regular and possibly rather dense molecular layer of various chemical species (molecules, nanoparticles, biomolecules, ligands for metal ions or anions, molecular receptors, oligomers or polymers, etc.) on conducting or semiconducting or non-conducting material surfaces. The method of the invention enables to control the density of functional groups present at the surface of the nanomaterials in order to introduce post-functionalization.
The calixarene layer can comprise various calixarenes.
The examples below make use of three types of calixarenes: a functional calixarene (C1), a functional PEGylated calixarene (C2) and a PEGylated calixarene (C3). The versatility of this procedure is even greater as the core can be made of metal alloys and the organic layer can be a mixed layer of calixarenes in order to combine their different properties.
The abbreviation NP or NPs designate nanoparticle(s).
The abbreviation LSPR refers to the Localized Plasmon Resonance band displayed by the metallic nanoparticles.
General Synthetic Scheme
Scheme 1 illustrates the general method of the invention to convert a mixture of at least one metal-based salt with at least one calix[4]arene diazonium salt (C1, C2 and/or C3) to obtain a metal-based nanomaterial with a core being pure metal or an alloy, coated with a calixarene layer, of either single type or combined type.
Scheme 2 shows the three calix[4]arenes that were used to make different layers, each possessing unique properties:
Mixed layers of these calix[4]arenes possess combination of these properties with the possibility to provide to the functionalized particles the desired properties by controlling the proportion of the calix[4]arenes within the mixed layer.
General Synthesis Procedure 1.
In a protein LoBind eppendorf, 150 μL of a 10 mM metal salt solution are mixed successively with 575 μL of milliQ water and 360 μL of a solution of a 5 mM calixarene milliQ water and the pH is adjusted to 6.5 using a NaOH solution (1M). Quickly after this, 410 μL of a 15 mM sodium ascorbate solution are added and the Eppendorf is placed overnight in a thermomixer at 60° C. and agitated at 800 RPM.
The sodium ascorbate is present at around 4 mM concentration in the reaction mixture.
After 16 hours, the nanoparticles are centrifuged (20 minutes at 18.000 g) and the supernatant is removed and replaced by around 1500 μL of a washing medium. This washing procedure is repeated four times. The medium replacing the supernatant depends on the calixarene used (table 1). In the case of silver nanoparticles, the final concentration of nanoparticles obtained after the washing step is evaluated by measuring the extinction coefficient of the silver particles following the method disclosed in D. Paramelle, A. Sadovoy, S. Gorelik, P. Free, J. Hobley and D. G. Fernig, A rapid method to estimate the concentration of citrate capped silver nanoparticles from UV-visible light spectra, Analyst, 2014, 139, 4855.
When the refractive index of a particle is known, the final concentration of particles can also be measured by DLS measurement (Malvern-Zetasizer) to give the concentration of particles (particles/mL).
Alternatively, the number of produced particles can be extrapolated from the quantity of reduced metal (the difference between the total quantity of metal added and the quantity of remaining cations in the solution, measured for example by ICP), the average size of the particles (obtained by TEM) and the density of the metal.
The UV-Visible spectrum of the coated nanomaterial is recorded using an absorption spectrometer. The coated nanomaterials are typically diluted 10 to 12 times in pure water for recording the UV-Visible spectra.
The colloidal stability is evaluated by comparing UV-Vis spectrum of the coated nanomaterial at pH 3, pH 7 and pH 12. Alteration of the UV-Visible spectrum at pH 3 is the sign of particles aggregation. Reversibility can be evaluated by assessing recovery of the UV-Vis spectrum when increasing the pH.
The chemical stability of the coated nanomaterial is evaluated by recording the UV-Visible spectra of the coated nanomaterial in solution at different time intervals after addition of a 150 mM potassium fluoride solution. No or little alteration of the UV-Visible spectrum means that the colloidal stability is not affected by the addition of KF, indicating that the metallic surface is not accessible to the fluoride ions, which is a sign of a dense and homogeneous repartition of the calixarenes on the surface of the metal-based core.
The presence of calixarenes on the metallic core is confirmed by IR spectroscopy.
The size and the shape of the metallic core of the particles was determined by analysis with SEM (scanning electron microscopy) and/or TEM (transmission electron microscopy).
The hydrodynamic diameter of the particles is analyzed by Dynamic Light Scattering (DLS) at a 100× dilution in water.
General observations for the experimental conditions:
Nanoparticles with pure silver core and coated with calixarene C1 were synthesized according to the general synthesis procedure 1, using silver nitrate as silver salt.
The UV-visible spectrum of Ag—C1-20 (
The final concentration of nanoparticles was estimated to be around 2.5 nM, based on a calculated extinction coefficient of 4.109 L.mol−l.cm−1, which is in good agreement with the extinction coefficient reported in the literature for particles of that size (20 nm as shown by TEM).
Colloidal stability is affected at low pH the Ag—C1-20 particles are aggregated as the carboxylic acids of C1 are protonated, increasing the initially negative surface potential of the particles, and decreasing the electrostatic repulsion between them. A typical spectrum of DLCA (Diffusion Limited Colloidal Aggregation) aggregated particles with a second absorption band around 490 nm is observed. However, this aggregation is reversible by increasing the pH thanks to the highly protective calixarene layer.
Chemical stability—After 12 hours of exposure to potassium fluoride, only few particles were degraded, meaning that the silver surface is hardly accessible to the fluoride thanks to the dense and strongly anchored calixarene layer (
The IR spectrum of Ag—C1-20 (
The hydrodynamic diameter of Ag—C1-20 diluted 100 times in milliQ water at 25° C. is around 35 nm
The synthesis was repeated at larger scales (1.5 mL, 20 mL, 30 mL of metal salt solution. A larger LSPR and a broader size distribution of articles was observed when increasing the scale to 20 mL and 30 mL. A further low speed centrifugation step (5000 g) was added at the end of the synthesis to remove the largest nanoparticles and narrow the LSPR.
Nanoparticles with pure silver core and coated with calixarene C1 were synthesized according to the general synthesis procedure 1 and example 1, but at three different temperatures: 20° C., 40° C. and 60° C.
The UV-visible spectrum (
Nanoparticles with pure silver core and coated with calixarene C1 were synthesized according to the general synthesis procedure 1 and example 1, but at various pH ranging between 3.0 and 12.0.
The UV-visible spectrum (
The chemical stability of commercially available silver nanoparticles, coated with citrate (Ag-Citrate, S-20-XX, Cytodiagnostics (Burlington, Canada)) or by a thiolated-PEG ended by a carboxylic acid (Ag-SPEG, SC3K-20-X*, Cytodiagnostics (Burlington, Canada)) was recorded for comparison under the same conditions as Ag—C1.
As shown on
In terms of shelf life, LSPR analysis of Ag—C1 showed no degradation after 29 months, while a small shift was observed after 4 months for Ag-citrate and almost full degradation was observed at 4 months for Ag-SPEG.
Nanoparticles with pure silver core and coated with calixarene C1 were synthesized according to the method disclosed in Troian-Gautier et al., Chemcomm, 2016, (used to prepare gold nanoparticles with the calix[4]arene C1 in acetonitrile at 0° C., through a reduction with NaBH4), using either acetonitrile or water as solvent. The protocol for washing the particles was either the one of Troian Gautier or the protocol of General procedure 1. The observations are gathered in the table below:
This experiment demonstrates the superiority of the method of the invention to prepare coated nanomaterials.
Nanoparticles with pure silver core and coated with calixarene C1 were synthesized according to the general synthesis procedure 1 using silver nitrate as silver salt, but using, instead of a final 4 mM sodium ascorbate concentration:
Nanoparticles with pure silver core and coated with calixarene C1 were synthesized according to the general synthesis procedure 1 using silver nitrate as silver salt, but pH is maintained at 3.5 (no addition of NaOH during the synthesis). The freshly synthesized silver nanoparticles were analyzed by UV-Vis spectroscopy. A strong and sharp LSPR band was observed with a maximum of absorbance at 430 nm, (
This was confirmed by DLS measurement that revealed a hydrodynamic diameter approximately 2.5 times bigger than the one of Ag—C1-20 prepared at pH7 (
The colloidal stability of these particles was similar to the one of 20 nm particles (
Calixarene C1 bears functional carboxylate groups, allowing additional chemical reactions (post-functionalization of the surface). Formation of an amide bond between carboxylates of Ag—C1 and ligands containing an amine group was demonstrated with NH2-PEG7-OCH3 (
Nanoparticles with pure silver core and coated with calixarene C2 were synthesized according to the general synthesis procedure 1, using silver nitrate as silver salt.
The UV-visible spectrum of Ag—C2 (
The final concentration of nanoparticles was estimated to be around 0.4 nM, based on a calculated extinction coefficient of 2.1010 L.mol−1.cm−1, which is in good agreement with the extinction coefficient reported in the literature for particles of that size (35 nm as shown by TEM).
Colloidal stability and chemical stability are affected by pH variation: during a pH variation cycle (neutral-acidic-basic) and upon KF exposure a loss of Ag—C2 particles is observed, both indicating a less efficient coating than in the case of Ag—C1 calixarenes (
The IR spectrum of Ag—C2 (
SEM and TEM showed spherical particles with a core size of around 35 nm (
In addition to interesting optical properties, the interest of silver cores lies in their antimicrobial activity. Despite the dense and stable coating of Ag—C1, these particles express similar or even greater antimicrobial activity than citrate-capped silver nanoparticles (Ag-Citrate).
E. Coli growth inhibition was studied by disc diffusion method for different conditions: 1=water; 2=Ag-citrate; 3=Ag—C1; 4=Ag—C2; 5=60 μM Kanamycine; 6=60 mM 30 Kanamycine).
From the test plate, it is clear that Ag—C1 and Ag—C2 are as efficient as Ag-Citrate in the case of the disc diffusion method and more efficient than 60 μM of Kanamycine, even if this well-known antibiotic is more than 1000 times more concentrated than the particles ([AgNPs]=0.6 nM and [Kanamycine]=60 μM).
In the case of the inhibition in solution, a stronger growth inhibition is observed with Ag—C1 than with Ag-Citrate, characterized by a lower optical density at 600 nm, after a defined incubation time, which could be explained by a longer lifetime of the Ag—C1 in the culture medium due to the protective calixarene-based layer. Ag—C2 is as efficient as Ag-Citrate.
Nanoparticles with pure silver core and coated with calixarene C3 were synthesized according to the general synthesis procedure 1, using silver nitrate as silver salt.
The UV-visible spectrum of Ag—C3 (
The final concentration of nanoparticles was estimated to be around 0.4 nM, based on a calculated extinction coefficient of 2.1010 L.mol−1.cm−1, which is in good agreement with the extinction coefficient reported in the literature for particles of that size (around 46 nm as shown by TEM).
Colloidal stability and chemical stability are only slightly affected by pH variations (
The IR spectrum of Ag—C3 (
SEM and TEM showed spherical particles with a core size of around 46 nm (
pH is important for obtaining optimal nanoparticles. The optimal pH for a narrow size distribution of Ag—C3 is 7. Deviation from this pH leads to larger size distribution.
Nanoparticles with pure silver core and coated with a mixture of calixarenes C1 and C2, in various ratio, were synthesized according to the general synthesis procedure 1.
The results are summarized in table 1 below.
The UV-visible spectra of the Ag—C1C2 (
The final nanoparticle concentrations were estimated to be around 0.4-2.5 nM, based on the calculated extinction coefficient.
Colloidal stability and chemical stability: The chemical robustness of these hybrid Ag—C1C2 particles is demonstrated by exposing the particles to extreme conditions, either acidic (
The IR spectrum of Ag—C1C2 (
Nanoparticles with pure silver core and coated with a mixture of calixarenes C1 and C3, in various ratio, were synthesized according to the general synthesis procedure 1.
The results are summarized in table 2 below.
The UV-visible spectra of the Ag—C1C3 (
The final concentration of nanoparticles was estimated to be around 0.14-2.5 nM, based on the calculated extinction coefficient.
Colloidal stability and chemical stability: The chemical robustness of these hybrid Ag—C1C3 particles is demonstrated by exposing the particles to extreme conditions, either acidic (
The IR spectrum of Ag—C1C3 (
Nanoparticles with a pure silver core and coated with a mixture of calixarenes C3 and C2, in various ratio, were synthesized according to the general synthesis procedure 1.
The results are summarized in table 3 below.
The UV-visible spectra of the Ag—C2C3 (
The final concentration of nanoparticles was estimated to be around 0.1-0.4 nM, based on the calculated extinction coefficient, depending on the amount of calixarene C2.
Colloidal stability and chemical stability: The chemical robustness of these hybrid Ag—C2C3 particles is demonstrated by exposing the particles to extreme conditions, either acidic or etching environment (
The IR spectrum of Ag—C2C3 (
Several calixarene coated gold nanomaterials or gold and silver coated nanomaterials were prepared using the same experimental procedure but starting with a gold salt: HAuCl4.
Data regarding UV and IR spectrum, stability studies are gathered in Table 4.
Overall, it was possible to product gold nanoparticles coated with a stable and homogeneous layer of calixarenes (single type or mixtures). These particles demonstrate high colloidal and chemical robustness and possess the capacity to be post-functionalized. Nanomaterials with a core comprising gold and silver were prepared, the ratio of metal incorporated in the core matching the ratio of the oxidized metal used for the synthesis. Similarly, to the silver nanomaterials described above, mixed layers of calixarenes could also be successfully be prepared, with a good control of the layer composition.
Nanoparticles with pure gold core and coated with calixarene C1 were synthesized according to the general synthesis procedure 1 using HAuCl4 as gold salt, but pH is maintained at 2 (no addition of NaOH during the synthesis). The freshly synthesized gold nanoparticles were analyzed by UV-Vis spectroscopy. A strong and sharp LSPR band was observed with a maximum of absorbance at 540 nm, (
This was confirmed by DLS measurement that revealed a hydrodynamic diameter approximately 2.5 times bigger than the one of Au—C1 prepared at pH7 (
The colloidal stability of these particles was similar to the one of 20 nm particles (
Several calixarene-coated palladium, platinum, copper oxide and iron oxide nanomaterials were prepared using general procedure 1, starting from the appropriate salts: palladium chloride, platinum chloride, copper acetate or mix of Iron Chloride salts (33% FeCl2/66% FeCl3)
These experiments show the versatility of the method of the invention, to make metallic cores coated with a monolayer of calixarenes. The metallic cores can be single metal, metal oxide or an alloy or metallic mixture. The ratio of the metals in the nanoparticle obtained is proportional to the ratio of oxidized metal introduced in the reaction mixture.
Depending on the metal, different shapes of nanoparticles can be obtained, in particular, nanospheres and nanocubes have been obtained.
The method of the invention works in water and in other solvents, like DMSO.
The method enables to obtain nanoparticles coated with several types of calixarenes, which is particularly convenient for introducing a variety of functionalities or post-functionalization possibilities. This can lead to advantageous properties in many different fields of activities, like microelectronics, ND, biomedical applications, catalysis, nanoreactors formed by calixarenes at the surface of particles, . . . .
The repeatability of the experiments, and stability of the nanoparticles enable to extrapolate the results of the above experiments to all types of calixarenes, and many more metals.
Silver based and gold based nanomaterials coated with calixarenes according to the method of the invention were used for preparing various immunoassays, in particular Ag—C1-20 as prepared in example 1, Ag—C2 as prepared in example 4, as well as Au—C1 and Au—C2 as prepared in example 10.
Unless otherwise stated, in the examples below, the anti-SARS-CoV-2 human IgG, and S-protein relate to Anti-Spike-RBD fully human mAb(IgG) and Recombinant SARS-CoV-2, S1 Subunit Protein (RBD) ordered from Tebu-Bio and Ray-Biotech respectively. An anti-SARS-CoV-2 human IgG and IgM mix was also ordered from MyBiosource.
Ag—C1-20 or Au—C1 was stirred in the presence of 100 eq./NP of Human Anti-IgG (Goat Anti-Human IgG (I2136) acquired from Sigma-Aldrich (Saint-Louis, Mo.)) or Human Anti-IgM (Goat Anti-Human IgM acquired from Sigma-Aldrich (Saint-Louis, Mo.)) (both 2 mg/mL in PBS 10 mM ) during 2 h at room temperature at 1000 rpm to provide Ag-C1-Anti-Human-IgG-ad and Ag-C1-Anti-Human-IgM-ad particles respectively. After 2 hours, the mixture was centrifuged at 18000 g for 20 minutes. The LSPR had shifted from 417 nm to 426 nm, indicating a change in dielectric environment. The supernatant was discarded, and the pellet was resuspended in ultrapure water. This operation was repeated three times in total. The resulting particles were further characterized by IR spectroscopy. The presence of intense amide I and II bands confirmed the adsorption of anti-Human-IgG on the silver nanoparticles (
Similarly, the shifts in the UV-visible spectra of the colloids confirmed the presence of Anti-human-IgG and Anti-human-IgM on the gold nanoparticles (
SARS-CoV-2 S protein was adsorbed onto calixarene coated silver particles with the aim to detect anti-SARS-CoV-2 human IgG in the blood of patients.
Protein-S was adsorbed on Ag—C1 based on adapted protocol found in literature. (ORCID iD: 0000-0001-8854-275X)
Three conditions were tested:
On the contrary to adsorption, bioconjugation involves creating at least one amide bond between a substituent of the calixarene coated to the surface of the metal nanomaterial and the protein (Immunoglobulin).
Attempts to bioconjugate anti-Rabbit IgG on silver nanoparticles stabilized with citrate, a thioalted PEG ended by a carboxylic acid (Ag-SPEG) or calix[4]arene were performed by activation of the carboxylic acid moieties with EDC/NHS. As can be seen from
The bioconjugation is performed as follows:
In a glass vial, 250 μL of Ag—C1-20 (final OD=1), 380 μL of ultrapure water, 200 μL of PBS (pH 7.4, final C=20 mM), 100 μL of Sulfo-NHS (final C=0.1 mM), 100 μL of EDC (final C=0.06 mM) and 20 μL of rabbit anti-IgG (0.2 mg/mL) were added. The suspension was then stirred at room temperature at 1000 rpm for 4 hours. After 4 hours, 0.5 mL of BSA 1% in PBS 20 mM was added to the suspension and centrifuged at 18000 g for 20 minutes. The supernatant was discarded, and the pellet was resuspended in 1.5 mL of bovine serum albumin (BSA) 1% in PBS 20 mM. This operation was repeated once more to yield Ag-C1-anti-Rabbit-IgG-pc.
These nanoparticles were characterized by UV-Vis, DLS and IR spectroscopies. DLS confirmed the increase in hydrodynamic diameter upon bioconjugation. Indeed, this diameter increased from 37 nm for Ag—C1 to 180 nm for Ag-C1-anti-rabbit-IgG-pc. Infrared spectroscopy allowed to highlight the presence of amide bands I and II of the anti-rabbit IgG after bioconjugation.
Similarly, Bioconjugation of anti-Rabbit-IgG on Au—C1 was performed using the same procedure to yield Au-C1-anti-Rabbit-IgG-pc.
Bioconjugation of anti-human-IgG was performed on Ag—C1-20 and Au—C1. These reactions were carried out either in water or in borate buffer. Overall, it seems that carrying the reaction in borate buffer allows to obtain better results and to lose less nanoparticles.
To 1 mL of Ag—C1-20 (concentration=0.5 nM) or Au—C1 (concentration=5 nM) dispersed in ultrapure water in a glass vial were added 100 μL sulfo-NHS (final C=1 mM), 100 μL of EDC, and 50 μL of anti-human-IgG (2 mg/mL, approximately 100 eq./NP). The reaction was stirred at room temperature at 1000 rpm for 16 hours. After reaction, the mixture was split in two series. Series A was washed three times with ultrapure water and series B was washed once with SDS 1% and twice with water. Each time, the mixture was centrifuged at 18000 g for 20 minutes. This procedure yielded either Ag-C1-anti-human-IgG-pc or Au-C1-anti-human-IgG-pc. No relevant difference was observed for the final products obtained with washing of Series A and series B.
Anti-Human-IgG were also bioconjugated to Ag—C2. The nanoparticles were activated via the EDC/NHS procedure and cleaned from excess reagent via centrifugation cycles. These activated nanoparticles were then reacted overnight with approximately 100 biomolecules per nanoparticle. The corresponding Ag-C2-Anti-Human-IgG-pc were purified via centrifugation cycles and resuspended in PBS 10 mM and stored in the fridge. Bioconjugation was confirmed by UV-Vis and IR spectroscopy (
Bioconjugation of anti-human-IgM was performed on Ag—C1-20 and Au—C1 as described above to yield Ag-C1-anti-human-IgM-pc and Au-C1-anti-human-IgM-pc. These reactions were carried out either in water or in borate buffer, as for example 16. Overall, it seems that carrying the reaction in borate buffer allows to obtain better results and to lose less nanoparticles. The obtained bioconjugated nanoparticles were characterized by UV-Vis spectroscopy as well as by IR spectroscopy. Amide bands I (1642 cm−1) and II (1527 cm−1) are clearly observable after bioconjugation.
Bioconjugation of the S1 subunit of the SARS-CoV-2 S-Protein was performed on Au—C1 as in examples 16 and 17. These bioconjugated nanoparticles were characterized by UV-Vis spectroscopy as well as by IR spectroscopy. Amide bands I (1652 cm−1) and II (1531 cm−1) are clearly observable after bioconjugation. This procedure yielded Au-C1-S1-pc.
Bioconjugation of SARS-CoV-2 S-Protein was also performed on Ag—C1 as in examples 16 and 17. This procedure yielded Ag-C1-S1-pc. Additional treatment of the particles was obtained using a blocking protein, casein, added to the Ag-C1-S1-pc particles. After incubation with casein, particles were cleaned by centrifugation to yield Ag-C1-S1-pc-casein particles.
Coupling of Protein S was also performed of Ag—C2. The carboxylates were activated in MES buffer via the EDC/NHS procedure and cleaned from excess reagent via centrifugation cycles. These activated nanoparticles were then reacted overnight with approximately 100 biomolecules per nanoparticle. The corresponding Ag-C2-S1-pc were purified via centrifugation cycles and resuspended in PBS 10 mM and stored in fridge. Bioconjugation was confirmed by UV-Vis and IR spectroscopy.
Rabbit-IgG solutions (1 mg/mL in PBS 10 mM, approximately 100 eq./NP; Rabbit IgG (I5006) acquired from Sigma-Aldrich (Saint-Louis, Mo.)), were added to solutions of Ag—C1-20 (0.24 nM) or Au—C1 (2.4 nM) suspended in PBS 50 mM at pH 7.4 to reach IgG concentrations varying from 0 to 220 nM. An UV-vis spectrum of each mixture was recorded. The ratio between the absorbance of aggregated nanoparticles ((λmax+175 nm) and the absorbance of dispersed nanoparticles (λmax) was reported to estimate the aggregation level.
In these conditions, Ag—C1-20 allowed to detect Rabbit-IgG concentrations as low as 1.6 nM while Au—C1 are unable to detect Rabbit IgG at concentrations lower than 20 nM (see
The adsorption (aggregation) process was shown to be reversible upon washing with sodium dodecyl sulfate (SDS).
Molecular sensing of human antibodies was performed. The analyte was a Human IgG capable of binding specifically the S1 subunit of the SARS-CoV-2 S-protein. Two batches of silver nanoparticles were used. One was bioconjugated with the subunit S1 of the SARS-CoV-2 S-protein (Ag—C2-S1-pc). The second batch was bioconjugated with an Anti-Human IgG, i.e. Ag-C2-Anti-Human-IgG-pc, capable of recognizing specifically Human IgGs. In the presence of anti-Sars-CoV-2 human IgG (Anti-Spike-RBD fully human IgG), the particles aggregate via the formation of a sandwich-type ternary complex, leading to a modification of their optical properties and thus the detection signal. A scheme of the procedure is represented in
The two sets of bioconjugated particles, i.e. Ag-C2-S1-pc and Ag-C2-Anti-Human-IgG-pc were mixed together in a quartz cuvette with 5 mM of PBS. Specific volumes of antibody solution were then added, and the UV-Vis spectrum was recorded after 10 minutes of incubation. IgG anti-SARS-CoV-2 (i.e. Anti-Spike-RBD fully human mAb(IgG) ). As a control, Rabbit-IgG was use. A linear increase of the absorbance at 600 nm, signature of aggregation of the particles, was observed when adding 1 nM to 4 nM of fully Human anti-SARS-CoV-2, but not with addition of Rabbit-IgG, even at concentration up to 250 nM.
In conclusion, this detection system allowed the specific detection of anti-Sars-Cov2 IgG with a low limit of detection at 1 nM.
Comparative experiments were run using Ag-citrate and Au-citrate nanomaterials, giving a much less sharp detection result.
Similar experiments were performed with Ag-C1-S1-pc and Ag-C1-Anti-Human IgG-pc, onto which anti-SARS-CoV-2 human IgG was added at increasing concentration (2 nM to 23.2 nM), giving a linear decrease and broadening of the LSPR.
These assays demonstrate the feasibility of a turbidimetry to detect immunoglobulins, and in particular anti-SARS-CoV-2 human IgG. These are very promising results towards the preparation of an immunoassay based on nanomaterials coated with calixarenes.
The examples below relate to the development of Lateral Flow Immunoassays (LFIA). In such an assay, the objective is to immobilize the functionalized nanoparticles on a strip in the presence of the analyte to detect, leading to the formation of a colored band that can be detected by the naked eye or a suitable detection technique. The sample to analyze is deposited on one extremity of the strip, combined with the functionalized nanoparticles and move towards the detection band by capillarity. If the analyte to be detected is present, it forms a bridge between the functionalized nanoparticles and the proteins/immunoglobins used to functionalize the strip at the level of the detection band, leading to the immobilization of the particles on this band and its coloration in red or yellow, depending on the use of AuNPs or AgNPs.
For development purpose, the assay is set up vertically, meaning that instead of depositing the analyte solution on an extremity of the strip, the extremity of the strip is plunged vertically in the analyte solution which moves up by capillarity along the strip.
Detection of anti-SARS-CoV-2 human IgGs was performed via a vertical flow assay involving strips functionalized at the level of the detection band with the protein A. The protein A is able to specifically bind to the heavy chains of IgG. The strips coated with proteins A and G were ordered from Abcam (Cambridge, United-Kingdom) as part of “Conjugation Check&Go” kit (ab236554).
In this context, anti-SARS-CoV-2 human IgGs can be immobilized on the detection band, and this interaction can be visually highlighted with engineered plasmonic nanoparticles (
1. Comparison Between Gold and Silver Nanoparticles
Calixarene-coated silver nanoparticles (Ag—C1-20) conjugated to Anti-Human IgG via adsorption (Ag-C1-Anti-Human-IgG-ad), as prepared in example 13, were compared to citrate-capped gold nanoparticles (Au-Citrate) conjugated to Anti-Human IgG via adsorption (Au-Citrate-Anti-Human-IgG_ad). In both cases, the particles can be immobilized on the strip via the interaction between the protein A and the Anti-Human IgG (
Control experiments were first performed to show the effect of the bioconjugation. It was confirmed that, in the absence of conjugation, Ag—C1 does not get immobilized on the strip.
Nb: All the Ag-C1-biomolecules used in this part come from the NPs batches cleaned with Casein and described above in “Nanoparticles Bioconjugation” part.
Strips, onto which a band of protein A and G was present, were vertically plunged into 100 μL of either Ag-C1-Anti-Human-IgG-ad-Casein suspensions, at concentrations ranging from 0.2 to 80 pM, or Au-Citrate-Anti-Human-IgG-ad-Casein suspensions, at concentrations ranging from 2 to 800 pM. After 20 minutes of incubation/migration the strips were removed from the suspensions and pictures were taken (
Ag-C1-Anti-Human-IgG-ad-Casein could be clearly observed on the strip for concentrations as low as 2 pM, and even detected at extremely low concentration (0.2 pM), with the naked eye. For Au-Citrate-Anti-Human-IgG-ad-Casein, concentration of 20 pM is the limit of observable signal with the naked eye and no coloration of the detection band could be observed at lower concentrations.
Ag-C1-Anti-Human-IgG-ad-Casein at the same concentration (20 pM) than gold nanoparticles lead to a much stronger signal thanks to their higher extinction coefficient.
2. Anti-Sars-Cov2 Human IgG Detection in Buffer
Ag—C1 conjugated with the SARS-CoV-2 S-protein via adsorption (Ag-C1-S1-ad-Casein) was used for the detection of anti-SARS-CoV-2 human IgG. Briefly, the strips, onto which a band of protein A and G has been deposited, were vertically incubated with 100 μL of solutions containing different concentrations of anti-SARS-CoV-2 human IgG (From 500 ng/mL to 1 ng/mL initially in PBS buffer) mixed with Ag-C1-S1-ad-Casein (initially in pure water) in buffer provided by the strip supplier, at an optical density of 0.1.
Strong detection signals could be observed down to concentration of 20 ng/mL of anti-SARS-CoV-2 human IgG. Concentration of 5 ng/mL could barely be observed by the naked eye and concentration of 1 ng/mL led to absolutely no signal. Control experiments confirmed the specificity of the interaction: (i) in the absence of anti-SARS-CoV-2 human IgG, no signal was observed and (ii) in the presence of Rabbit-IgG, no signal was observed.
In order to enhance the detection signal for concentration of 5 ng/mL, a higher concentration of silver nanoparticles was used (OD=0.4). The signal obtained was stronger than the one obtained with an OD of 0.1 Optimizing the concentration of nanoparticles appears to help to obtain a detectable signal at low IgG concentration.
Another strategy investigated was to use Ag—C1 conjugated to the viral S-protein via peptide coupling (Ag-C1-S1-pc-Casein). No significant difference was observed with this conjugation strategy compared with bioconjugation by adsorption
3. Anti-Sars-Cov2 Human IgG Detection in Pure Buffer with Ag—C2
Ag—C2 conjugated with the SARS-CoV-2 S-protein (Ag-C2-S1-pc) and Anti-human IgG (Ag-C2-Anti-Human-IgG-pc), both via amide bond formation, were used for the detection of anti-SARS-CoV-2 human IgG. Strips were vertically incubated with 100 μL of solution containing 200 ng/mL of anti-SARS-CoV-2 human IgG mixed with either Ag-C2-S1-pc, Ag-C2-Anti-Human-IgG-pc or both at an optical density of 0.1.
No signal was observed for the Ag-C2-Anti-Human-IgG-pc only, probably due to the saturation of the particles by the different IgGs present in the sample, preventing any interaction with the strip. In the other hand, Ag-C2-S1-pc allowed the visual detection of anti-SARS-CoV-2 human IgG.
An even stronger signal was obtained with the sandwich strategy involving the simultaneous presence of the two bioconjugated nanoparticles. This may be explained by the immobilization of nanoparticle aggregates on the strip instead of single particles (
The signal intensity with serum containing different amount of anti-SARS-CoV-2 human IgG: 5, 2.5, 1.5, 1, 0 μg/mL were compared.
10 μL of serum (containing different concentrations of IgG), 10 μL of Ag-C1-S-Prot-Casein-ad (OD=5), 5 μL of Rabbit IgG-AuNPs (provided by Abcam) for control line and an appropriate amount of running buffer to reach a total volume of 50 μL were used to dip therein strips onto which a band of Protein A is present.
A signal can be easily read on the strip for serum containing 5 and 2.5 μg/mL of SARS-Cov-2 IgG. For serum containing 1.5 μg/mL, a poorly discernible signal can also be observed. No signal can be observed for serum containing 1 and 0 μg/mL.
3 different batches of nanoparticles with different coatings and bioconjugation strategies were compared: one type where the proteins are adsorbed onto the calixarene C1 coated nanoparticles Ag-C1-S1-ad-Casein, and two types where the proteins are bioconjugated by peptide coupling to the calixarene coated nanoparticles of Ag-C1-S1-pc-Casein and Ag-C2-S1-pc. The volumes of NPs solutions were adjusted in order to have similar OD in the different assay suspensions.
All NPs were tested with serum containing 2.5, 1.5, 1.0 and 0 μg/mL of anti-SARS-CoV-2 human IgG. Overall, peptide coupled NPs (pc) allowed to improve the limit of detection, giving a reliable signal at 1.5 μg/mL and weak signal at 1.0 μg/mL.
These results confirm the better bioconjugation through amide coupling compared to the mere chemical adsorption.
It was tested to deposit, on a same strip, two parallel lines, one composed of anti-human IgG and one composed of anti-human IgM. When a sample containing a mix of both the anti-SARS-CoV-2 human IgG and IgM incubated with Ag-C1-S1-pc-Casein in the presence of 10 μL serum (containing 1.5 μg/mL IgG and IgM mix) was used, both bands became visible.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20156678.3 | Feb 2020 | EP | regional |
| 20205365.8 | Nov 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/053170 | 2/10/2021 | WO |
