Nanoparticles complexed with functionalizable enhanced affinity ligands and use thereof

Abstract

Disclosed are functionalizable ligands, nanoparticles, preferably nanocrystals, complexed with ligands and their use for bio-imaging. A nano material includes a nanoparticle and at least one copolymer ligand. A ligand which is a copolymer of general formula (I): HP[(A)x-co-(B)y]n-L-R.

Claims

1. A nanomaterial comprising: a nanoparticle; and at least one ligand which is a copolymer of formula (I):
H-Poly[(A).sub.x-co-(B).sub.y].sub.n-L-R wherein H represents a hydrogen atom; co means copolymer; Poly[(A).sub.x-co-(B).sub.y].sub.n designates a copolymer with a total of n monomers, a percentage x of said n monomers is monomer A and a percentage y of said n monomers is monomer B, wherein: monomer A represents an anchoring monomer having a side-chain comprising a first moiety M.sub.A having affinity for the surface of a nanocrystal comprising a metal; monomer B represents a hydrophilic monomer having a side-chain comprising a second moiety M.sub.B being hydrophilic; n represents a positive integer; x and y are different from 0% of n and different from 100% of n; wherein x+y is equal to 100% of n; R represents: a functional group selected from the group consisting of NH.sub.2, COOH, OH, SH, CHO, ketone, halide, activated ester, activated carboxylic acid, isothiocyanate, isocyanate, alkyne, azide, glutaric anhydride, succinic anhydride, maleic anhydride; hydrazide; chloroformate, maleimide, alkene, silane, hydrazone, oxime and furan; or a bioactive group selected from the group consisting of avidin, streptavidin, antibody, sugars, and a protein or peptide sequence having a specific binding affinity for an affinity target; and L represents a spacer which is an ethylene glycol derivative of formula [CH.sub.2CH.sub.2O]n.sub.2-(CH.sub.2)n.sub.3-S wherein n.sub.2 and n.sub.3 are each independently positive integers and n.sub.2+n.sub.3 is higher than 2 and wherein L is linked to Poly[(A).sub.x-co-(B).sub.y].sub.n through the S atom of [CH.sub.2CH.sub.2O]n.sub.2-(CH.sub.2)n.sub.3-S group.

2. The nanomaterial according to claim 1, wherein said nanoparticle is a nanocrystal and wherein the nanocrystal is a 0D, 1D, or 2D nanocrystal.

3. The nanomaterial according to claim 1, wherein said nanoparticle is selected from the group consisting of a nanosheet, a nanorod, a nanoplatelet, a nanoplate, a nanoprism, a nanowall, a nanodisk, a nanoparticle, a nanowire, a nanopowder, a nanotube, a nanotetrapod, a nanoribbon, a nanobelt, a nanoneedle, a nanocube, a nanoball, a nanocoil, a nanocone, a nanopiller, a nanoflower, and a quantum dot.

4. The nanomaterial according to claim 1 wherein the ligand is of formula (II): ##STR00037## wherein co, n, x, y, L and R are as defined in claim 1; R.sub.A represents a group comprising the first moiety M.sub.A having affinity for the surface of a nanocrystal comprising a metal; R.sub.B represents a group comprising the second moiety M.sub.B being hydrophilic; R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 represent each independently H or a group selected from the group consisting of alkyl, alkenyl, aryl, hydroxyl, halogen, alkoxy, carboxylate, and amide.

5. The nanomaterial according to claim 1, wherein the ligand is of formula (I-e): ##STR00038## wherein co, n, x, y, L and R are as defined in claim 1.

6. The nanomaterial according to claim 1, wherein the ligand is of formula (I-f): ##STR00039## wherein co, n, x, y and L are as defined in claim 1.

7. A water-soluble composition comprising nanomaterials comprising: a nanoparticle; and at least one ligand which is a copolymer of formula (I):
H-Poly[(A).sub.x-co-(B).sub.y].sub.n-L-R wherein H represents a hydrogen atom; co means copolymer; Poly[(A).sub.x-co-(B).sub.y].sub.n designates a copolymer with a total of n monomers, a percentage x of said n monomers is monomer A and a percentage y of said n monomers is monomer B, wherein: monomer A represents an anchoring monomer having a side-chain comprising a first moiety M.sub.A having affinity for the surface of a nanocrystal comprising a metal; monomer B represents a hydrophilic monomer having a side-chain comprising a second moiety M.sub.B being hydrophilic; n represents a positive integer; x and y are different from 0% of n and different from 100% of n; wherein x+y is equal to 100% of n; R represents: a functional group selected from the group consisting of NH.sub.2, COOH, OH, SH, CHO, ketone, halide, activated ester, activated carboxylic acid, isothiocyanate, isocyanate, alkyne, azide, glutaric anhydride, succinic anhydride, maleic anhydride, hydrazide, chloroformate, maleimide, alkene, silane, hydrazone, oxime and furan; or a bioactive group selected from the group consisting of avidin, streptavidin, antibody, sugars, and a protein or peptide sequence having a specific binding affinity for an affinity target; and L represents a spacer which is an ethylene glycol derivative of formula [CH.sub.2CH.sub.2O]n.sub.2-(CH.sub.2)n.sub.3-S wherein n.sub.2 and n.sub.3 are each independently positive integers and n.sub.2+n.sub.3 is higher than 2 and wherein L is linked to Poly[(A).sub.x-co-(B).sub.y].sub.n through the S atom of [CH.sub.2CH.sub.2O]n.sub.2-(CH.sub.2)n.sub.3-S group.

8. A method for manufacturing the nanomaterial according to claim 1 comprising: a first step of complexation of nanocrystals with an intermediate ligand being a weakly binding ligand or a small molecule ensuring the homogeneous dispersion of the nanocrystal into a solvent miscible in part with water; a step of monophasic exchange at about 40 C. to about 100 C. in an aqueous medium overnight to remove the weak intermediate ligand and replace it by the ligand which is a copolymer of general formula (I):
H-Poly[(A).sub.x-co-(B).sub.y].sub.n-L-R wherein H represents a hydrogen atom; co means copolymer; Poly[(A).sub.x-co-(B).sub.y].sub.n designates a copolymer with a total of n monomers, a percentage x of said n monomers is monomer A and a percentage y of said n monomers is monomer B, wherein: monomer A represents an anchoring monomer having a side-chain comprising a first moiety M.sub.A having affinity for the surface of a nanocrystal comprising a metal; monomer B represents a hydrophilic monomer having a side-chain comprising a second moiety M.sub.B being hydrophilic; n represents a positive integer; x and y are different from 0% of n and different from 100% of n, wherein x+y is equal to 100% of n; R represents: a functional group selected from the group consisting of NH.sub.2, COOH, OH, SH, CHO, ketone, halide, activated ester, activated carboxylic acid, isothiocyanate, isocyanate, alkyne, azide, glutaric anhydride, succinic anhydride, maleic anhydride, hydrazide, chloroformate, maleimide, alkene, silane, hydrazone, oxime and furan; or a bioactive group selected from the group consisting of avidin or streptavidin; antibody; sugars; and a protein or peptide sequence having a specific binding affinity for an affinity target; and L represents a spacer which is an ethylene glycol derivative of formula [CH.sub.2CH.sub.2O]n.sub.2-(CH.sub.2)n.sub.3-S wherein n.sub.2 and n.sub.3 are each independently positive integers and n.sub.2+n.sub.3 is higher than 2 and wherein L is linked to Poly[(A).sub.x-co-(B).sub.y].sub.n through the S atom of [CH.sub.2CH.sub.2O]n.sub.2-(CH.sub.2)n.sub.3-S group.

9. The nanomaterial according to claim 1, wherein the at least one ligand is a copolymer of general formula (III): ##STR00040## wherein co, L and R are as defined in claim 1, R.sub.A and R.sub.A represent respectively a group comprising a first moiety M.sub.A and a group comprising a first moiety M.sub.A, said moieties M.sub.A and M.sub.A having affinity for the surface of a nanocrystal comprising a metal; R.sub.B and R.sub.B represent respectively a group comprising a second moiety M.sub.B and a group comprising a second moiety M.sub.B, said moieties M.sub.B and M.sub.B being hydrophilic; R.sup.1, R.sup.2 R.sup.3 R.sup.4, R.sup.5, R.sup.6, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 represent each independently H or a group selected from the group consisting of alkyl, alkenyl, aryl, hydroxyl, halogen, alkoxy, carboxylate, and amide; n represents a positive integer; x and x represent each independently a percentage of n, wherein at least one of x and x is different from 0% of n; wherein x and x are different from 100% of n; y and y represent each independently a percentage of n, wherein at least one of y and y is different from 0% of n; wherein y and y are different from 100% of n; and wherein x+x+y+y is equal to 100% of n.

10. The nanomaterial according to claim 9, wherein x and/or y is equal to 0.

11. The nanomaterial according to claim 9, wherein M.sub.A and M.sub.A are each independently selected from an imidazole moiety and a carboxylic acid or carboxylate moiety, and M.sub.B and M.sub.B are each independently selected from a group with both an ammonium group and a sulfonate group, a sulfobetaine group, a PEG, a poly(ether)glycol moiety, a carboxybetaine moiety wherein the ammonium group may be included in a five-membered heterocycle comprising 1, 2 or 3 further nitrogen atoms, a sulfobetaine moiety wherein the ammonium group may be included in a five-membered heterocycle comprising 1, 2 or 3 further nitrogen atoms and a phosphobetaine wherein the ammonium group may be included in a five-membered heterocycle comprising 1, 2 or 3 further nitrogen atoms.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A and 1B are .sup.1H NMR spectra recorded in D.sub.2O showing the monomer consumption during the polymer synthesis, between the initial state of the polymerization (FIG. 1A) and the end of the polymerization (FIG. 1B).

(2) FIGS. 2A et 2B are photographs of coated dots QD-PEG-SA, QD-SA and QD-PEG-COOH observed without any apparatus after reaction with biotin-coated agarose beads.

(3) FIG. 2C is a photograph of coated dots QD-PEG-SA, QD-SA and QD-PEG-COOH observed by microscopy after reaction with biotin-coated agarose beads.

EXAMPLES

(4) The present invention will be better understood with reference to the following examples. These examples are intended to representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.

Abbreviations

(5) AIBN: azobisisobutyronitrile;

(6) APMA.HCl: N-(3-aminopropyl)methacrylamide hydrochloride;

(7) CTA: Chain Transfer Agent;

(8) DCC: dicyclohexylcarbodiimide;

(9) EDC: 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride;

(10) LA: 5-(1,2-dithiolan-3-yl)pentanoic acid, also known as lipoic acid;

(11) MES buffer: 2-(N-morpholino)ethanesulfonic acid aqueous buffer;

(12) MFA: N-methylformamide;

(13) MPA: 3-mercaptopropionic acid;

(14) NHS: N-Hydroxysuccinimide;

(15) PEG: poly(ethylene glycol);

(16) QD(s): Quantum Dot(s);

(17) SA: streptavidin;

(18) SPP: 3-sulfopropyldimethyl-3-methacrylamidopropylammonium inner salt.

Materials and Instrumentation

(19) Streptavidin was purchased from Biospa; APMA.HCl was purchased from Tebu-bio; SPP and 3-[3-methacrylamidopropyl-(dimethyl)ammonio]propane-1-sulfonate), from Raschig GmbH (RaleMer SPP); all other chemicals used therein were purchased from Sigma-Aldrich. All of these purchased chemicals were used without further purification unless otherwise specified. Chromatography on silica was carried out on Kieselgel 60 (230-240 mesh, Merck) and analytical TLC was performed on Merck precoated silica gel (60 F.sub.254) .sup.1H NMR spectrum was recorded on a Bruker Avance DPX 400 spectrometer at 400.13 MHz. Chemical shifts () are expressed in ppm and coupling constant (J) in hertz. Absorption measurements were carried out with a Cary 5E UV-vis-NIR spectrophotometer (Varian).

Example 1

Ligand Synthesis

(20) Synthesis of monomer A (5-(1,2-dithiolan-3-yl)-N-(3-methacrylamidopropyl)-pentanamide)

(21) ##STR00031##

(22) To a suspension of APMA.HCl (2 g, 11.2 mmol) in dichloromethane (20 mL) was added triethylamine (2.5 mL, 17.9 mmol). Methanol (2 mL) was introduced to obtain complete solubilization. A solution of LA (2.76 g, 13.4 mmol) in dichloromethane (5 mL) was then added, followed by NHS (1.58 g, 13.8 mmol) in one portion. The reaction mixture was cooled down to 0 C. with an ice bath and a solution of DCC (3.00 g, 14.4 mmol) in dichloromethane (10 mL) was injected dropwise. The medium was warmed up to room temperature and further stirred overnight. A pale yellow solution containing a white precipitate was obtained. The solution was washed by a 0.1 M aqueous HCl solution (250 mL), deionized water (150 mL) and a 0.2 M aqueous NaOH solution (250 mL). The organic phase was separated, dried over MgSO.sub.4, filtrated and concentrated under reduced pressure. The crude residue was purified by chromatography on silica (eluent: hexane/ethyl acetate 1/4, then hexane/acetone 1/1) to give A (2.88 g, 8.71 mmol, 78%) as a pale yellow solid. R.sub.1=0.37 (hexane/acetone 1/1); .sup.1H NMR (CDCl.sub.3, 400 MHz): 7.03 (sl, 1H); 6.87 (sl, 1H); 5.72 (s, 1H); 5.29 (s, 1H); 3.53-3.39 (m, 1H); 3.29-3.20 (m, 4H); 3.14-3.01 (m, 2H); 2.43-2.35 (m, 1H); 2.18 (t, J=8.0 Hz, 2H); 1.92 (s, 3H); 1.88-1.80 (m, 1H); 1.68-1.55 (m, 6H); 1.48-1.33 (m, 2H).

(23) Synthesis of the Ligand

(24) The polymerization step consisted of the radical copolymerization of two methacrylamides: one containing the precursor of a dithiol anchoring function (monomer A, obtained as described above), the other including a sulfobetaine group (monomer B, SPP commercially available).

(25) Due to mismatching monomers' solubilities, the solvent used for these polymerizations was acetic acid. Various A/B molar ratios have been tested between 10/90 and 50/50.

(26) The amounts of initiating agent AIBN (2,2-Azobis(2-methylpropionitrile)) and of CTA were chosen in various molar equivalents relative to the total amount of monomers, in order to form various length chains. Various monomer/CTA molar ratios have been tested between 10/1 and 40/1. The monomers conversion rates were determined by .sup.1H NMR in D.sub.2O (classically over 90%).

(27) Characterization of the products by NMR confirmed the M.sub.A/M.sub.B molar ratio by comparison of the acrylamide peaks between the initial state of the polymerization (FIG. 1A) and the end of the polymerization (FIG. 1B).

(28) ##STR00032##
A general procedure is given for the synthesis a ligand with x=2 and n=10.

(29) Monomer B (SPP, 1.17 g, 4 mmol, 4 equiv.), monomer A (5-(1,2-dithiolan-3-yl)-N-(3-methacrylamidopropyl)pentanamide, 331 mg, 1 mmol, 1 equiv.) and CTA (0.5 mmol, 0.5 equiv.) were dissolved in acetic acid (20 mL) in a sealed septum flask. The mixture was degassed with argon and then heated at 60 C. A solution of AIBN (82 mg, 0.5 mmol, 0.5 equiv.) in acetic acid (2 mL) under argon atmosphere was further added in one portion. The mixture was stirred overnight at 60 C. Acetic acid was evaporated under reduced pressure. To remove residual acetic acid traces, the residue was dissolved in 20 mL of deionized water and evaporated under reduced pressure (two times). The residue was then dissolved in 20 mL of deionized water and extracted three times with 20 mL of dichloromethane. The aqueous phase was precipitated in 9-fold excess of ethanol. The precipitated polymer was separated by centrifugation (50 mL centrifuge tubes, 2500 rpm, 10 min), washed 2 times with ethanol and then dried overnight under vacuum. The polymer was obtained as a yellow-brown solid (950 mg, 61%).

(30) TABLE-US-00006 Chain Transfer Agents (CTA) 0.5 mmol Structures Mercaptopropionic acid (MPA) 42 L Cysteamine 38 mg O-(2-carboxyethyl)- O-(2-mercaptoethyl) heptaethylene glycol 229 mg

(31) In the case wherein the ligand is obtained by polymerization with mercapto-carboxilic acid containing compounds as CTA, the ligand is referred to as ligandCOOH. Especially, in the case wherein the ligand is obtained by polymerization with mercaptoproprionic acid as CTA, the ligand is referred to as ligand-(CH.sub.2).sub.2COOH and in the case wherein the ligand is obtained by polymerization with a CTA comprising a PEG moiety, the ligand is referred to as ligand-PEG-COOH.

(32) In the case wherein the ligand is obtained by polymerization with mercapto-amino containing compounds as CTA, the ligand is referred to as ligandNH.sub.2.

Example 2

Quantum Dot Synthesis

(33) CdSe/CdS/ZnS QDs Synthesis

(34) 600-nm-emitting CdSe/CdS/ZnS QDs were synthesized using slight modifications of previously published procedures. CdSe cores were synthesized by reaction of trioctylphosphine selenide and cadmium oleate in octadecene, oleylamine and trioctylphosphine oxide. Three monolayers of CdS shell, followed by two monolayers of ZnS, were grown using cadmium oleate, zinc oleate and sulfur diluted in octadecene following the SILAR (Successive Ionic Layer Adsorption and Reaction) procedure.

Example 3

Nanocrystal Complexation

(35) Ligand Exchange: Standard Procedure

(36) Classical biphasic cap exchange with CdSe/CdS/ZnS core/shell QDs solubilized in chloroform did not succeed. The poor solubility of the ligand of the invention in chloroform and the low partition coefficient between the two solvents could explain the difficult phase transfer of the QDs.

(37) To overcome this problem, a two-step process was chosen. A first exchange was performed using pure mercaptopropionic acid (MPA), on as-synthesized QDs precipitated in ethanol. The QDs were kept overnight at 60 C. in order to have QDs surface was saturated by MPA. The excess of MPA was removed and QDs were dispersed in DMF. The MPA were then deprotonated using a large excess of tert-butoxide. The QDs became then instable in organic solvents and were precipitated and washed with ethanol. QDs were then suspended in a sodium tetraborate (pH 9, 10 mM) water-based buffer. To this homogeneous dispersion, an aqueous solution of previously reduced ligand of the invention (by NaBH.sub.4) was added to perform the second ligand exchange. The aqueous medium was kept overnight at 60 C. to move from the weak intermediate QD covered by MPA to QD-ligand. The polymer excess was removed by Vivaspin filtration. The QD-ligand did not show any change in quantum yield after the ligand exchange.

(38) General Procedure

(39) CdSe/CdS/ZnS core/shell QDs in hexane (0.2-2 nmol respectively for 650-550 nm QDs) were precipitated with ethanol (0.5 mL) and centrifuged (13000 rpm, 5 min). The supernatant was removed. The QDs were dispersed in 3-mercaptopropionic acid (MPA) (0.2 mL). The mixture was sonicated to obtain a homogenous dispersion. The QDs dispersion was stored at 60 C. overnight to perform first cap exchange. The QDs were centrifuged (13000 rpm, 2 min) and the MPA phase was discarded. The QDs were dispersed in DMF (0.2 mL) under sonication. 2 mg of potassium tert-butoxide were added and QDs dispersion was sonicated (1 min). The mixture was centrifuged (13000 rpm, 2 min). The uncolored DMF phase was discarded. The precipitated QDs were washed twice with ethanol (20.5 mL EtOH). The QDs were dispersed in sodium tetraborate (pH9 10 mM). Typically, at this step, QDs colloidal dispersion was clear. 200 L of aqueous solution of the ligand of the invention (10 mg/mL), previously reduced 30 min with NaBH.sub.4 (1 mg/mg of polymer), were added to QDs dispersion. The aqueous QDs dispersion was stored at 60 C. overnight to perform the second cap exchange. The excess of free ligand and reagents were removed by three washing by membrane ultrafiltration (Sartorius Vivaspin500 L disposable filtercutoff 100 kDa) at 13000 rpm in 20 mM aqueous NaCl. QDs-ligand were finally taken up in 20 mM aqueous NaCl.

(40) In the case wherein the ligand is a ligandCOOH, as in example 1, resulting coated QDs are referred to as QDs-ligandCOOH.

Example 4

Activation of the Ligand on the Nanocrystal

(41) In the case wherein the ligand of the invention is obtained by polymerization with mercapto-carboxilic acid containing compounds as CTA (for example: mercaptopropionic acid or O-(2-Carboxyethyl)-O-(2-mercaptoethyl)heptaethylene glycol), an acidic function is advantageously introduced at one extremity of the ligand (respectively ligand(CH.sub.2).sub.2COOH and ligand-PEG-COOH).

(42) Ligand exchange using these ligandCOOH has been studied in order to provide biocompatible coated QDs presenting carboxylic acidic function, QDs-ligandCOOH. First, reactive N-hydroxysuccinimide (NHS) esters thereof (QD-ligandNHS) were prepared and purified. Then, these activated QDs have been used to functionalize QDs with protein (for example: streptavidin or antibodies).

(43) Washing Before Freeze-Drying: Standard Procedure

(44) QDs-ligandCOOH in 20 mM aqueous NaCl were washed three times by membrane ultrafiltration at 13000 rpm using a Sartorius Vivaspin500 L disposable filter (cutoff 100 kDa) in pure water. QDs-ligandCOOH were finally taken up in pure water for freeze-drying.

(45) Activation of the Carboxylic Acidic Function of the Polymer on QDs: Standard Procedure

(46) Freeze-dried QD-ligandCOOH (2.5 mg) were dispersed in pure water (50 L) at room temperature. In parallel, EDC (5 mg, 30 mol) and NHS (5 mg, 44 mol) were dissolved in MES buffer 0.2 M pH 5.5 (1 mL). 20 L of this solution are added to the dispersion of QD and immediately after 180 L of MFA are added. The reaction was stirred overnight at room temperature before precipitation in acetonitrile (1 mL). The colored precipitate obtained after centrifugation (13000 rpm, 2 min) was washed twice in acetonitrile (1 mL) before drying under vacuum. QDs-ligandNHS were conserved under inert atmosphere at 18 C.

(47) ##STR00036##
Biomolecules Functionalization of QDs:

(48) Several experiments were carried out starting from either QDs-ligand-COOH or QDs-ligand-NHS.

(49) QDs-ligand-(CH.sub.2).sub.2COOH represents quantum dots coated with a ligand copolymer wherein the end carboxylic function is from 3-mercaptopropionic acid. QDs-ligand-(CH.sub.2).sub.2NHS correspond to the corresponding dots after the reaction with N-hydroxysuccinimide.

(50) QDs-ligand-PEG-COOH represents quantum dots coated with a ligand copolymer wherein the end carboxylic function is from a CTA comprising a PEG moiety; especially from (2-carboxyethyl)-O-(2-mercaptoethyl)heptaethylene glycol. QDs-ligand-PEG-NHS corresponds to the corresponding dots after the reaction with N-hydroxysuccinimide.

(51) Standard Procedure from QDs-ligandNHS

(52) Dried QDs-ligandNHS (2.5 mg) were dispersed in 120 L streptavidine or antibody solution (10 mg/mL in aqueous NaHCO.sub.3 0.2 M pH 8.4). Protein excess was eliminated by ultracentrifugation on sucrose gradient (40%-10%). QDs-proteins/QDs-antibody were finally taken up in 0.2M aqueous NaHCO.sub.3.

(53) Characterizations Biotin Test

(54) First the quantum dots functionalized by streptavidin were tested with biotin-coated agarose beads in order to evaluate the achievement of the functionalization. The results are shown Table 1 and FIGS. 2A, 2B and 2C.

(55) TABLE-US-00007 TABLE 1 Results of the test on biotin-coated agarose beads. Reaction with biotin- coated Reagent 1 Reagent 2 Obtained agarose (coated quantum dots) (protein-NH.sub.2) product beads* QDs-ligand-PEG-COOH Streptavidin QDs-PEG-SA + QDs-ligand- Streptavidin QDs-SA 0 (CH.sub.2).sub.2COOH QDs-ligand-PEG-COOH QDs-PEG-COON 0 QDs-ligand- QDs-(CH.sub.2).sub.2COOH 0 (CH.sub.2).sub.2COOH *+: positive result; 0: negative result

(56) In this test, the high affinity between streptavidin and biotin leads to fluorescence.

(57) The results of Table 1 show that when quantum dots are coated with a ligand comprising a PEG moiety (QDs-ligand-PEG-COOH), their functionalization with streptavidin leads to fluorescent agarose beads (+).

(58) In the case wherein quantum dots are coated with a ligand comprising a S(CH.sub.2).sub.2COOH end obtained from mercaptopropionic acid (QDs-ligand-(CH.sub.2).sub.2COOH), the reaction between the resulting quantum dots and the agarose beads does not lead to fluorescent beads.

(59) These results show that when the ligand is ended by S(CH.sub.2).sub.2COOH, the functionalization with streptavidin fails whereas in the case wherein the ligand comprises a PEG moiety, the functionalization of the QDs-PEG-COOH is successfully achieved.

(60) Without willing to be bound by a theory, the Applicant thinks that the size of the spacer affects the functionalization of the coated-quantum dots. The skilled human in the art knows that carboxylic function has good affinity for the surface of dots. Thus, increasing the size of the spacer L would have favored the coiling of the end of the ligand chain on the dots surface, preventing the functionalization by a protein, a fluorophore or an antibody.

(61) Unexpectedly, these experiments show that an enough long spacer is required to implement the functionalization of the QDs-NHS with a protein or an antibody.

(62) Number of Fluorophores by Quantum Dot

(63) Functionalized quantum dots were analyzed by HPLC with an absorbance detector at 254 nm or a fluorescence detector (emission parameters: L.sub.exc.=630 nm and LF=650 nm). The number of fluorophores grafted on a quantum dots is calculated by the ratio between the absorption band of QDs (at 350 nm) and the absorption band of fluorophores (at 650 nm).

(64) The results are shown in the following table:

(65) TABLE-US-00008 Functionalized QDs Number of fluorophores (F) by QD QDs-PEG-F 0.5 QDs-F 0

(66) These results show that when quantum dots are coated with a ligand comprising a -PEG-COOH moiety and then functionalized with a fluorophore (QDs-PEG-F), the functionalization reaction is successfully carried out and the average number of flurophores per dot is 0.5.

(67) In the case wherein quantum dots are coated with a ligand comprising a (CH2)2-COOH end and then functionalized with a fluorophore (QDs-F), the reaction of functionalization fails.

(68) In conclusion, these results confirm that the design of the ligand end is important. Especially, a too short spacer L does not allow the functionalization of the ligand-coated QDs.