EP2350229A1 - Fluorescent nanoparticles, method for preparing same, and application thereof in biological marking - Google Patents

Abstract

The present invention relates to a method for preparing nanocrystals including a semiconducting ternary compound that comprises the elements A, B and C, and more particularly a semiconductor of the formula ABC2 optionally coated with a shell having an external portion that includes a semiconductor of the formula ZnS1-xFx, with A being a metal or a metalloid at the +I oxidation state, B being a metal or a metalloid at the +III oxidation state, C being an element at the -II oxidation state, F being an element at the -II oxidation state, and x being a decimal number such that 0 = x < 1. The present invention also relates to the nanocrystals thus prepared and to the uses thereof.

Description

 FLUORESCENT NANOPARTICLES, PROCESS FOR PREPARING THEM AND THEIR APPLICATION IN BIOLOGICAL MARKING

DESCRIPTION

TECHNICAL AREA

The present invention belongs to the technical field of semiconductor nanocrystals.

More particularly, the present invention proposes a method for preparing nanocrystals comprising a semiconductor of ternary composition, typically of type ABC2, such as nanocrystals of CuInS2 optionally coated with a layer of another type DE semiconductor such as ZnS. The present invention also relates to the novel luminescent materials thus prepared and in particular based on CuInS2 / ZnS core / shell nanocrystals whose emission covers the visible spectrum and the near infrared as well as their various uses and in particular for in vivo imaging.

STATE OF THE PRIOR ART

Two main types of Near-Infrared Fluorescent Markers (or "labels" in English), which is the wavelength range for which light absorption and light scattering by biological tissues are minimal, have been developed for in vivo imaging. These markers are either molecular fluorescent markers, ie organic fluorophores absorbing / emitting in the near infrared, or nano-particle fluorescent markers.

The only organic fluorophore today authorized for injection in humans is indocyanine green (or ICG for "IndoCyanine Green"). Unfortunately, this fluorophore is poorly soluble in aqueous buffer, has no chemical group allowing its grafting to a biological targeting ligand, such as a peptide, an antibody or a protein, and is strongly adsorbed on plasma proteins. Clinical applications using ICG are, at present, essentially applications in vascularization imaging, such as angiography of the eye. This is why many other cyanines absorbing / emitting in the near infrared have been developed and patented in recent years (Amersham, Molecular Probes, Schering, Li-Cor, Biosearch), the efforts made to develop such cyanines on improving their solubility and their functionalization by grafting groups enabling them to be coupled to biomolecules probes of biological processes, such as peptides. These fluorophores, some of which are now marketed at very high prices, are not currently known and approved for injection into humans, although toxicity studies in this direction are most certainly underway at several suppliers. Some of these organic fluorophores are however used for fluorescence imaging of small animals. So the Visen company commercializes various probes based on organic fluorophores to visualize in the small animal the blood flow, glucose consumption of tumors, osteoblastic activity, the activity of metalloproteases, the activity of cathepsins and plasmin.

However, these fluorophores have the drawbacks inherent to the organic fluorophores absorbing / emitting in the near infrared: high photobleaching speed, wide emission lines, low spectral shift between absorption and emission, and low quantum yields of emission (10 to 15% in water for fluorophores emitting above 650 nm). For these reasons, semiconductor nanocrystals have quickly emerged as an interesting alternative as luminescent markers for biology. Semiconductor crystals are luminescent materials known for several decades. In the years 1980-1990, it was shown that their emission spectrum depends on the size of the crystal when it becomes sufficiently small. For crystals whose size is approximately in the range of 1 to 10 nm called "nanocrystals" or "quantum boxes", this dependence is extremely pronounced. In fact, the entire palette of colors of the visible and near infrared and ultraviolet can be obtained with semiconductor nanocrystals by the appropriate choice of their size and composition. To cover the visible and near-infrared spectrum, very Important in the fields of lighting / display and biological labeling, the most studied materials are chalcogenides of cadmium (CdS, CdSe, CdTe) and lead (PbS, PbSe). However, the European RoHS Directive (Restriction of Hazardous Substances) provides for the elimination of, among others, lead, mercury and cadmium in Electrical and Electronic Equipment (EEE), marketed in Europe from 1 July 2006. Also, due to their intrinsic toxicity, nanocrystals based on cadmium [1], lead or arsenic [2] are not acceptable for a large number of applications as a biological marker, and in particular for in vivo labeling in the body human. It is therefore essential to find alternative materials for the production of nanocrystals, while keeping the desired optical properties.

In general, the optical quality of a luminescent material composed of nanocrystals depends on several parameters, the most important of which are:

- the size of the nanocrystals, which regulates the emission wavelength; - the size distribution of the nanocrystals, which controls the emission linewidth; the surface passivation of the nanocrystals, responsible for the fluorescence quantum yield and stability over time. Roquesite of formula CuInS2 (or CIS), a member of the ternary chalcopyrite family, is an interesting material for substituting cadmium and lead nanocrystals. Thanks to its bandwidth of 1.5 eV, it is possible to vary the emission wavelength of CIS nanocrystals in the visible spectrum and in the near infrared by changing their size. However, CIS nanocrystals generally show very low fluorescence efficiency at room temperature, as evidenced by the literature.

There are several methods for preparing CIS nanocrystals, among which the method of decomposing precursors in a high temperature organic solvent gives access to the narrowest size dispersion. A small size dispersion of the nanocrystals leads to a narrow emission spectrum, i.e. a pure emission color which is particularly advantageous for technological applications. The main synthesis routes are as follows.

A first synthetic route was described in 2003 in the article by Castro et al. [3]. The latter have proposed the use of (PPh ₃ ) ₂ CuIn (SEt) ₄ which is a monomolecular precursor, at the same time a source of copper, indium and sulfur. This precursor is placed in a flask containing as solvent dioctyl phthalate. The mixture, heated to 200 ^° C., leads to the formation of a red powder. This powder is isolated, purified, dispersed in dioctyl phthalate and then heated to 250-300 ^° C. A brown or black powder consisting of CIS nanocrystals is then obtained. A modification of this protocol includes the addition of hexanethiol to the reaction mixture and gives access to nanoparticles soluble in organic solvent [4]. The nanocrystals of CIS obtained have a size of between 2 and 4 nm and the maximum fluorescence quantum yield is around 5%. Another variant of the method consists in triggering the reaction by UV irradiation (photolysis) [5]. The main disadvantage of these approaches is the use of a monomolecular precursor which is not commercially available and which must therefore be synthesized beforehand using, for example, the procedure described in the article by Banger et al., 2003 [ 6].

Nakamura et al. have more recently described a second synthetic route that uses individual sources of copper, indium and sulfur [7]. In this case, the octadecene which serves as the solvent is mixed with copper iodide and indium iodide dissolved in oleylamine and sulfur, dissolved in trioctylphosphine. After heating the mixture at 160-240 ^° C., the size of the nanocrystals obtained is 3.5-7.5 nm, respectively. However, the fluorescence quantum yield is low, less than 0.1%. A value of 5% was obtained with the quaternary system, Zn-Cu-In-S, described in the same article.

Another very recent synthetic route uses copper and indium diethyldithiocarbamates prepared beforehand by the reaction of copper (II) chloride and indium (III) chloride. with sodium diethyldithiocarbamate [8]. Both precursors are mixed with oleic acid or dodecanethiol (stabilizer) in 1-octadecene

(solvent). After heating the reaction mixture at 200 ^° C., the oleylamine is injected, triggering the reaction to form CIS nanocrystals with a size of 4-30 nm according to the parameters chosen. No information concerning the photoluminescence properties of these crystals is given. In summary, the methods of preparation described above make it possible to obtain CIS nanocrystals in a wide range of sizes and with a small size dispersion. However, they do not solve the problem of fluorescence quantum yield which remains low (less than 5%). As a result, the intended applications of CIS nanocrystals are limited to solar cells, which are based on the nanoparticle absorption properties and not on their fluorescence. In the state of the art, the use of CIS nanocrystals as emitter / fluorophores and in particular in biological labeling is not described.

DISCLOSURE OF THE INVENTION The present invention makes it possible to solve the technical problems and disadvantages previously listed.

Indeed, the present invention proposes, first of all, a new process for the synthesis of nanocrystals comprising a semiconducting semiconductor compound consisting of elements A, B and C, hereinafter called ternary compound (A, B, C), and particularly in a stoichiometric form of formula ABC2. This method, completed by a step of coating with a shell, the external part of which comprises another semiconductor of formula DE, makes it possible to obtain nanocrystals, in particular nanocrystals of the "core / shell" type and, in particular, core nanocrystals. shell / formula CIS / ZnS having a fluorescence quantum yield greater than 10% and a high stability against photo-oxidation. In addition, the processes according to the present invention used are very simple to implement, which facilitates the change of scale to manufacture these nanocrystals in larger quantities. Finally, the method of the present invention is remarkable because not only it allows the preparation of CIS nanocrystals optionally coated with a ZnS shell, but also it is generalizable to the preparation of nanocrystals comprising a ternary compound (A, B, C) semiconductor, and particularly of formula ABC2, optionally coated with a shell comprising at least one semiconductor of formula DE with A representing a metal or metalloid in the oxidation state +1, B representing a metal or metalloid in the state oxidation agent + III, C representing an element in the oxidation state -II, D representing a metal or metalloid in the oxidation state +11 and E representing an element in the oxidation state -II. Thus, the present invention relates to a process for preparing a nanocrystal comprising a semiconducting ternary compound consisting of elements

A, B and C, and, more particularly, of formula ABC2, with A representing a metal or metalloid in oxidation state +1, B representing a metal or metalloid in oxidation state + III and C representing an element in the oxidation state -II, said process (hereinafter referred to as process (I)) comprising the successive steps of: a) preparing a mixture comprising at least one precursor of A, at least one precursor of B and at least one precursor of C at a temperature T _a ; b) maintaining the mixture obtained in step (a) at a temperature T _b greater than or equal to the temperature T _a ; c) bringing the mixture obtained in step (b) of the temperature T _b to a temperature T _c greater than the temperature T _b ; di) optionally purifying the nanocrystals comprising a ternary semiconductor compound consisting of elements A, B and C, and more particularly of formula ABC2, obtained in step

(vs) .

The nanocrystal prepared according to the method of the invention comprises a semiconducting semiconducting compound consisting of elements A, B and C, and more particularly of formula ABC2, with A representing a metal or metalloid in oxidation state +1, B representing a metal or metalloid in the state oxidation agent + III and C representing an element in the oxidation state -II, this type of semiconductor being called I-III-VI.

A, the metal or metalloid in the oxidation state +1 implemented in the context of the present invention is advantageously chosen between copper (Cu), silver (Ag) and mixtures thereof.

B, the metal or metalloid in the oxidation state + III used in the context of the present invention is advantageously chosen from gallium (Ga), indium (In), aluminum (Al) and their mixtures.

C, the element in the oxidation state -II used in the context of the present invention is advantageously chosen from sulfur (S), oxygen (O), selenium (Se), tellurium ( Te) and their mixtures.

By way of example of a ternary compound (A, B, C) semiconductor included in the nanocrystal prepared according to the process of the invention, there may be mentioned CuIn ₃ Se ₅ , CuIn ₃ S ₅ , CuIn ₅ SeS, CuIn ₅ Ss, Cu ₂ In ₂ Se ₄ , CuIn ₇ SeH ... Among the stoichiometric ternary compounds (A, B, C), there are typically the semiconductors of formula ABC ₂ , among which mention may in particular be made CuAlS ₂ , CuGaS ₂ , CuInS ₂ , CuAlO ₂ , CuGaO ₂ , CuInO ₂ , CuAlSe ₂ , CuGaSe ₂ , CuInSe ₂ , CuAlTe ₂ , CuGaTe ₂ , CuInTe ₂ , AgAlS ₂ , AgGaS ₂ , AgInS ₂ , AgAlO ₂ , AgGaO ₂ , AgInO ₂ , AgAlSe ₂ , AgGaSe ₂ , AgInSe ₂ , AgAlTe ₂ , AgGaTe ₂ , AgInTe ₂ and mixtures thereof. In particular, the core of the nanocrystal prepared according to the process of the invention comprises a semiconductor of formula ABC ₂ chosen from CuInS ₂ , CuGaS2, CuInSθ2, CuGaSθ2 and mixtures thereof, and more particularly, said semiconductor is CuInS2.

Advantageously, the nanocrystal prepared according to the method of the invention consists exclusively of a semiconductor as previously defined (ie a ternary semiconductor consisting of elements A, B and C and, more particularly, a semiconductor of formula ABC2 ).

The nanocrystal prepared by the process (1) of the present invention has a diameter of less than 10 nm, in particular less than 8 nm and in particular between 1 and 6 nm.

In the context of the present invention, the precursor of A used is selected from the group consisting of a copper precursor, a silver precursor, and mixtures thereof. All the precursors of copper and silver known to those skilled in the art and in particular the precursors in liquid or solid form can be used in the present invention.

Advantageously, the precursor of A is chosen from the salts of A, the halides of A, the oxides of A and the organometallic compounds of A. By "organometallic compound of A" is meant, more particularly, a compound of A substituted, a carboxylate of A or a phosphonate of A.

By "substituted A compound" is meant in the context of the present invention a compound of formula R1A in which R1 represents a hydrocarbon group of 1 to 20 carbon atoms such that a alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical.

By "A carboxylate" is meant in the context of the present invention a compound of formula R 2 COOA in which R 2 represents a hydrocarbon group of 1 to 20 carbon atoms such as an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical.

By "phosphonate of A" is meant in the context of the present invention a compound of formula R ₃ -P (OR ₄ ) (OR ₅ ) OA in which:

- R ₃ represents a hydrocarbon group of 1 to 20 carbon atoms such as an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical;

R ₄ represents a hydrogen atom or a hydrocarbon group of 1 to 20 carbon atoms such as an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical, and R ₅ represents a hydrogen atom; hydrogen or a hydrocarbon group of 1 to 20 carbon atoms such as an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical.

In the context of the present invention and unless otherwise indicated, the term "alkyl group" means a linear, branched or cyclic alkyl group, optionally substituted, of 1 to 20 carbon atoms, in particular of 1 to 15 carbon atoms, and in particular, from 1 to 10 carbon atoms. In the context of the present invention, the term "alkenyl group" means an alkenyl group. linear, branched or cyclic, optionally substituted, from 2 to 20 carbon atoms, in particular from 2 to 15 carbon atoms and, in particular, from 2 to 10 carbon atoms. In the context of the present invention, the term "alkoxy group" means an oxygen atom substituted with an alkyl as defined above.

In the context of the present invention, the term "aryl group" means an optionally substituted mono- or polycyclic aromatic group having from 6 to 20 carbon atoms, especially from 6 to 14 carbon atoms, in particular from 6 to 8 carbon atoms.

In the context of the present invention, the term "aryloxy group" means an oxygen atom substituted with an aryl as defined above.

In the context of the present invention, the term "optionally substituted" is a radical substituted with one or more groups chosen from: an alkyl group, an alkoxy group, a halogen, a hydroxy, a cyano, a trifluoromethyl or a nitro.

In the context of the present invention, the term "halogen" means a fluorine, chlorine, bromine or iodine.

As examples of the precursor of A that may be used in the context of the present invention, when A is copper, mention may be made of copper chloride, copper iodide, copper acetate and copper acetylacetonate, copper stearate, copper palmitate, copper myristate, copper laurate, copper oleate and mixtures thereof. More particularly, said copper precursor is copper iodide.

In the context of the present invention, the B precursor used is advantageously chosen from the group consisting of an indium precursor, a gallium precursor, an aluminum precursor and their mixtures. All the precursors of indium, aluminum and gallium known to those skilled in the art and in particular the precursors in liquid or solid form can be used in the present invention.

Advantageously, the precursor of B is chosen from among the salts of B, the halides of B, the oxides of B and the organometallic compounds of B. By "organometallic compound of B" is meant, more particularly, a compound of B trimer. substituted, a carboxylate of B or a phosphonate of B.

By "tri-substituted B compound" is meant in the context of the present invention a compound of formula (R 8) B in which each Re, identical or different, represents a hydrocarbon group of 1 to 20 carbon atoms such as an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical.

For the purposes of the present invention, the term "B carboxylate" means a compound of formula (R ₇ COO) 3B in which each R ₇ , which may be identical or different, represents a hydrocarbon group of 1 to 20 carbon atoms, such as an alkyl radical, a radical alkenyl, an alkoxy radical, an aryl radical or an aryloxy radical.

In the context of the present invention, the term "B phosphonate" means a compound of formula [R ₈ -P (OR ₉ ) (ORio) O] 3B in which: each Rg, which may be identical or different, represents a hydrocarbon group of 1 to 20 carbon atoms such as an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical; each Rg, which may be identical or different, represents a hydrogen atom or a hydrocarbon group of 1 to 20 carbon atoms such as an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical, and each Rio , identical or different, represents a hydrogen atom or a hydrocarbon group of 1 to 20 carbon atoms such as an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical.

The alkyl, alkenyl, alkoxy, aryl and aryloxy radicals are as defined for the precursors of A.

As examples of precursor of B usable in the context of the present invention, when B is indium, mention may be made of indium trichloride, triethylindium, indium triacetate and tri (acetylacetonate). indium, indium trioctanoate, indium tristearate, indium trilaurate, indium tripalmitate, indium trimyristate, indium trioleate and mixtures thereof. More particularly, said indium precursor is indium acetate.

In the context of the present invention, the C precursor used is selected from the group consisting of a sulfur precursor, an oxygen precursor, a selenium precursor, a tellurium precursor and mixtures thereof. All the precursors of sulfur, oxygen, selenium and tellurium known to those skilled in the art and in particular the precursors in liquid or solid form can be used in the present invention.

The precursor of C is selected from elemental selenium dissolved in an organic solvent; elemental tellurium dissolved in an organic solvent; elemental sulfur dissolved in an organic solvent; an aliphatic thiol; a xanthate; an amine oxide; a phosphine selenide; a phosphine oxide; a compound of formula C (Si (Rn) 3) 2 in which C represents a member selected from the group consisting of S, Se and Te and each Rn, which is the same or different, is a linear, branched or cyclic alkyl group, optionally substituted , from 1 to 10 carbon atoms, especially from 1 to 6 carbon atoms and, in particular, from 1 to 3 carbon atoms, and mixtures thereof.

Advantageously, the aliphatic thiol is of formula C _n H _{2n +} i-SH with n representing an integer between 1 and 25, in particular between 5 and 20 and, in particular, between 8 and 18. As thiols aliphatic agents that may be used in the context of the present invention include octanethiol (n = 8), octadecanethiol (n = 18), dodecanethiol (n = 12) and mixtures thereof. By "xanthate" is meant in the context of the present invention a compound of sequence

(Ri2 <0CS2) nY with Y representing a metal or metalloid in oxidation state + n and R12 representing a hydrocarbon group of 1 to 20 carbon atoms such as an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical. The alkyl, alkenyl, alkoxy, aryl and aryloxy radicals are as defined for the precursors of A.

By "amine oxide" is meant in the context of the present invention a compound of sequence (R13) 3NO in which each R13, identical or different, represents a hydrocarbon group of 1 to 20 carbon atoms such as an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical. The alkyl, alkenyl, alkoxy, aryl and aryloxy radicals are as defined for the precursors of A. As amine oxide that may be used in the context of the present invention, mention may be made of trimethylamine N-oxide (R13 = methyl). Advantageously, the organic solvent in which selenium, tellurium or elemental sulfur is dissolved is chosen from trialkylphosphines in which the alkyl group comprises from 4 to 12 carbon atoms and alkenes. By way of examples of organic solvents which may be used, mention may be made of 1-octadecene, tributylphosphine and trioctylphosphine.

More particularly, the selenide and oxide phosphines that may be used in the context of the present invention are respectively chosen from trialkylphosphine selenide and trialkylphosphine oxide in which the alkyl group comprises from 4 to 12 carbon atoms.

The precursors of A, B and C used in the context of the present invention may be commercially available products or products for which the person skilled in the art knows at least one simple method of preparation. Thus, at least one of the precursors of A, B and C may have been optionally prepared beforehand before being introduced into the mixture of step (a) or may be prepared in situ in said mixture.

The mixture of at least one precursor of A, at least one precursor of B and at least one precursor of C, ie the mixture prepared in step (a) of the process of the invention, is carried out in an organic solvent.

Advantageously, said organic solvent is an alkane, a secondary or tertiary amine, or an alkene having a boiling point greater than T _c , ie greater than the temperature chosen for step (c) of the process according to the invention.

By "alkane" is meant, in the context of the present invention, a linear, branched or cyclic alkane, optionally substituted, of 1 to 40 carbon atoms. carbon, especially from 10 to 35 carbon atoms and, in particular, from 14 to 30 carbon atoms. By way of example, the alkanes that may be used in the context of the present invention are hexadecane and squalane (C30H62)

By "secondary or tertiary amine" is meant, in the context of the present invention, especially dialkylamines and trialkylamines whose alkyl group comprises from 4 to 24 carbon atoms, especially from 8 to 20 carbon atoms. By way of example, the secondary amine (tertiary) which may be used in the context of the present invention is dioctylamine (trioctylamine) which has 8 carbon atoms per alkyl chain. By "alkene" is meant, in the context of the present invention, a linear, branched or cyclic alkene, optionally substituted, of 2 to 40 carbon atoms, in particular of 10 to 35 carbon atoms and, in particular, of 14 to 30 carbon atoms. at 40 carbon atoms. By way of example, an alkene that can be used in the context of the present invention is squalene (C30H50).

A solvent more particularly used to prepare the mixture of precursors in the process according to the invention is 1-octadecene (CiSH ₃₆ ).

In addition, the mixture of precursors in said solvent may further contain an element selected from the group consisting of a stabilizer for the surface of the nanocrystals and a primary amine. Indeed, the mixture of precursors in said solvent may further contain a stabilizer for the surface of the nanocrystals. By "stabilizer for the surface of the nanocrystals", also called "stabilizing ligand" means in the context of the present invention an organic molecule which binds to the surface of the nanocrystal and which thus avoids the aggregation of the nanocrystals. Any stabilizer known to those skilled in the art can be used in the context of the present invention. Advantageously, said stabilizer is chosen from thiols and especially aliphatic thiols as previously described; alkylphosphines and especially tri (alkyl) phosphines as previously described; alkylphosphine oxides and phosphonic acids as previously described; carboxylic acids and especially aliphatic or olefinic carboxylic acids such as lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid or mixtures thereof.

Finally, the mixture of precursors in said solvent may also contain a primary amine. Advantageously, the primary amine is an alkylamine whose alkyl group comprises from 4 to 24 carbon atoms, especially from 8 to 20 carbon atoms. By way of example, the primary amines which may be used in the context of the present invention are octylamine, dodecylamine, hexadecylamine (HDA) and oleylamine.

The concentration of the precursor of A, the precursor of B and the precursor of C in the mixture during step (a) of the process is between 2.5 and 150 mmol / L, especially between 5 and 100 mmol / L and in particular between 10 and 20 mmol / L.

It should be noted that some of the previously listed stabilizers are also sources of precursors used in the process according to the invention and in particular precursors of C such as sulfur precursors. In this case, it is clear that the total amount of a precursor also acting as a stabilizer is much greater in the mixture than the quantity defined above. Thus, the amount of a compound acting as precursor and stabilizer is between 10 mmol / L and 5 mol / L, in particular between 100 mmol / L and 1 mol / L and in particular between 200 and 700 mmol / L. .

Advantageously, the temperature T _a of the precursor mixture during step (a) is less than 50 ^° C., in particular less than 40 ^° C. and, in particular, less than 30 ^° C. More particularly, the mixture of precursors is at room temperature. By "ambient temperature" is meant a temperature of 20 ° C ± 5 ° C.

Advantageously, step (a) of the process of the invention is carried out with stirring. Various means known to those skilled in the art can be used to stir the mixture used in step (a) of the process of the invention. By way of example, the mixture can be stirred using a stirrer, a magnetic bar, an ultrasonic bath or a homogenizer. Finally, step (a) of the process of the invention may be carried out under a flow of an inert gas and in particular under a stream of argon, nitrogen or a mixture thereof.

Step (b) of the process of the invention aims at keeping the mixture prepared in step (a) at a temperature T _b greater than or equal to the initial temperature of the mixture, ie the temperature T _a as defined above. During this step the precursors of A and B can be converted by reaction with the stabilizer molecules in the medium. For example, indium triacetate can react with myristic acid to form indium trimyristate.

Advantageously, the temperature T _b of the precursor mixture during step (b) is less than 100 ^° C., in particular between 30 and 80 ^° C., in particular between 40 and 60 ^° C., more particularly, the mixture precursors is at a temperature of the order of 50 ^° C. "Temperature of the order of 50 ^° C." means a temperature of 50 ^° C. ± 5 ° C.

In a first variant of step (b) of the process according to the invention, the temperature T _b is equal to the initial temperature of the mixture, ie the temperature T _a of step (a). In this case, once the precursor mixture has been produced, the latter is maintained at the temperature T _b = T _a for a duration of between 15 and 180 min, in particular between 30 and 120 min and, in particular, for a period of between 60 min. By "duration of the order of 60 min" means a duration of 60 min ± 10 min.

In a second variant of step (b) of the process according to the invention, the temperature T _b is greater than the initial temperature of the mixture, ie the temperature T _a of step (a). In this case, once the mixture of precursors made, the latter is raised from the temperature T _a to T _b. This passage can be made increasingly linear or with at least one level. Particularly advantageously, this passage is increasingly linear, in particular with a ramp of 1 to 20 ^° C. per second, in particular a ramp of 2.5 to 15 ° C. per second and, more particularly, a ramp of 5 to 10 ° C. ⁰ C per second.

Step (b) is advantageously carried out under a primary vacuum. After step (b), purging with an inert gas such as argon, nitrogen or a mixture thereof is performed. Similarly, step (b) of the process of the invention is carried out with stirring and in particular according to the embodiments previously envisaged for step (a) of the process.

Step (c) of the process of the invention consists in gradually heating the reaction mixture obtained in step (b) of the process of the invention of the temperature T _b to a higher temperature, ie the temperature T _c . During this step, the formation and growth of the nanocrystals of the semiconducting ternary compound (A, B, C) take place. Advantageously, the temperature T _c is greater than 150 ^° C., in particular greater than 180 ^° C., in particular between 180 ^° C. and 300 ^° C., and more particularly between 200 ^° C. and 270 ^° C. Advantageously, the temperature T _c is of the order of 230 ° C. By "of the order of 230 ° C" means a temperature of 230 ^° C. ± 20 ^° C. and in particular a temperature of 230 ° C. ± 10 ^° C.

In a first variant of step (c) of the process according to the invention, the transition from the temperature T _b to the temperature T _c is increasingly linear.

Advantageously, the linear increase in temperature is carried out with a ramp of 0.5 to 20 ^° C. per second, in particular a ramp of 1 to 10 ^° C. per second and, more particularly, a ramp of 1.5 to 5 °. C per second.

In a second variant of step (c) of the process according to the invention, the transition from the temperature T _b to the temperature T _c is increasing with at least one step.

"Bearing" means a temperature T between T _b and T _c which is kept constant for a time between 5 sec and 1 h, in particular between 15 sec and 45 min, in particular between 30 sec and 30 min and, more particularly, between 1 min and 15 min. The increase in temperature between the temperatures T _b and T _c can have from 1 to 10 steps and in particular from 2 to 5 steps. Between T _b and the first step, between two consecutive levels and between the last step and T _c , the temperature is increased by linearly under the conditions as defined for the first variant of step (c).

In step (c), once the temperature T _{c has been} reached, this temperature can be kept constant for a period of between 10 minutes and 5 hours, in particular between 20 minutes and 3.5 hours and, in particular, between 30 minutes and 30 minutes. min and 2 h. Advantageously, the duration of step (c) is of the order of 40 min. By "duration of the order of 40 min" is meant a duration of 40 min ± 10 min and in particular a duration of 40 min ± 5 min.

Those skilled in the art know different techniques and different means for progressively bringing the precursor mixture of the temperature T _a to the temperature T _b and the temperature T _b to the temperature T _c . By way of examples, mention may be made of the use of a balloon or reactor containing the precursor mixture, programmable thermostatic, or the use of a previously heated bath at the required temperature which may be the temperature of a bearing, the temperature T _b or the temperature T _c in which is immersed the balloon or the reactor containing the mixture of precursors.

Step (c) is advantageously carried out with stirring and in particular according to the embodiments previously envisaged for step (a) of the process.

During step (di) of the process according to the invention, the nanocrystals obtained are purified from the reaction mixture, ie the nanocrystals are separated from said reaction mixture. Those skilled in the art know different techniques for this purification using a precipitation step, a dilution step and / or a filtration step. The techniques used in the prior art for purifying luminescent nanocrystals can be used in the context of step (d1) of the process of the invention.

Advantageously, the step (di) of the process is carried out at a temperature below the temperature T _c . Thus, the reaction mixture obtained after step (c) of the process is cooled or allowed to cool to room temperature. Next, the nanocrystals are purified by precipitation using a suitable solvent or mixture of solvents.

For example, a mixture of an alcohol such as methanol and chloroform (advantageously in a proportion of 1: 1 vol: vol) can be used to dilute the reaction mixture obtained following the step

(c) up to double volume, and then precipitate with excess acetone. The nanocrystals are recovered by centrifugation and can then be dispersed in organic solvents such as hexane, toluene or chloroform. The purification step (di) of the process according to the invention may optionally be repeated one or more times.

It is clear to those skilled in the art that there are two main ways to control the size of nanocrystals of ternary composition (A, B, C) obtained by the process (1) according to the invention and consequently their emission color. These main means are (1) the reaction time and (2) the adjustment of the reaction parameters. Those skilled in the art will therefore be able to adapt, without inventive effort, these parameters to obtain a nanocrystal of ternary composition (A, B, C) having a particular emission color.

The process (1) according to the present invention makes it possible to obtain nanocrystals of ternary composition (A, B, C) and particularly of formula ABC2 exhibiting a maximum observed fluorescence quantum yield. By way of example, when the nanocrystal prepared according to the method of the present invention is a CIS nanocrystal, the maximum fluorescence quantum yield observed is of the order of 8%.

A method widely used to increase the quantum yield of nanocrystals of various materials (eg CdSe, CdS, etc.) is to passivate their surface by growing around the nanocrystal corresponding to the "core" a shell of a semiconductor width of upper bandgap. This system is called "heart / shell" in the scientific literature. The methods used for the deposition of the shell are essentially the same as those used for the preparation of the heart. To improve the properties of nanocrystals of ternary composition (A, B, C), the present invention proposes to cover them with a shell of a DE semiconductor, D being a metal or metalloid in the oxidation state +11 and E representing an element in the oxidation state -II.

The present invention thus proposes a process for preparing a nanocrystal having (i) a core comprising a semiconductor of ternary composition (A, B, C), and more particularly of formula ABC2, with A representing a metal or metalloid in the oxidation state +1, B representing a metal or metalloid in the oxidation state + III and C representing an element in the oxidation state -II, coated by (ii) a shell whose outer portion comprises a semiconductor of the formula DE with D representing a metal or metalloid in the oxidation state + 11 and E representing an element in the oxidation state -II. A semiconductor of formula DE is also called II-VI. More particularly, the semiconductor implemented in the outer part of the shell has the formula ZnSi_ _x F _x , with F representing an element in the oxidation state -II and x being a decimal number such that 0 ≤ x <1 .

Thus, the present invention provides a process for preparing a nanocrystal having (i) a core comprising a semiconductor of ternary composition (A, B, C), and more particularly of formula ABC2, with A representing a metal or metalloid in oxidation state +1, B representing a metal or metalloid in oxidation state + III and C representing an element in oxidation state -II, coated by (ii) a shell of which the external part comprises a semiconductor of formula ZnSi_ _x F _x , with F representing an element in the oxidation state -II and x being a decimal number such that 0 ≤ x <1, said method (hereinafter referred to as process (2)) comprising the steps of: α) preparing a nanocrystal comprising a ternary semiconductor compound consisting of elements A, B and C, and more particularly of formula ABC2, according to a process as defined above, then β) coating the nanocrystal prepared in step (α) with a shell whose external portion comprises a semiconductor of formula ZnSi_ _x F _x , with F representing an element in the oxidation state -II and x being a decimal number such that 0 ≤ x <1.

The nanocrystal prepared by the process (2) of the present invention has a diameter of less than 15 nm, especially less than 12 nm and in particular less than 10 nm.

The shell of the nanocrystal prepared by the process (2) according to the invention has a thickness of between 0.3 and 6 nm, in particular between 0.5 and 4 nm and in particular between 1 and 2 nm.

Any technique known to those skilled in the art for coating or surrounding a nanocrystal of a semiconductor with one or more layers of another (or other) semiconductor (s) can be used in the frame of step (β) of the method according to the present invention. Said step (β) may be implemented on the nanocrystals prepared by the method (1), purified (ie following step (di)) or not (ie following step (c)). Advantageously, the process (2) according to the present invention comprises the successive steps of: a) preparing a mixture comprising at least one precursor of A, at least one precursor of B and at least one precursor of C at a temperature T _a ; b) maintaining the mixture obtained in step (a) at a temperature T _b greater than or equal to the temperature T _a ; c) bringing the mixture obtained in step (b) of the temperature T _b to a temperature T _c greater than the temperature T _b ; d2) adding, to the mixture obtained in step (c) and maintained at temperature T _c , at least one zinc precursor, at least one sulfur precursor and optionally at least one precursor of F; e2) purifying the nanocrystals having a core comprising a semiconducting ternary compound consisting of elements A, B and C, and, more particularly, of formula ABC2, coated with a shell whose outer layer comprises a semiconductor of formula ZnSi_ _x F _x , obtained in step (d2).

The various forms of implementation and variants envisaged for steps (a), (b) and (c) of the process (1) according to the invention apply mutatis mutandis to steps (a), (b) and (c). ) of the process (2) according to the invention.

The nanocrystal prepared according to the process (2) of the present invention has a shell whose external part comprises a semiconductor of formula ZnSi _x F _x , with F representing an element in the oxidation state -II and x being a decimal number such that 0 ≤ x <1. F is an element in the oxidation state -II chosen in particular from oxygen (0), selenium (Se), tellurium (Te) and mixtures thereof.

The shell of the nanocrystal prepared according to the method (2) of the invention may consist of a single layer or of several layers (i.e. be a multilayer shell). In the case where the shell consists of only one layer, the outer portion of the shell corresponds to said layer.

In the case where the shell consists of several layers, the outer part of the shell corresponds to the outer layer of the shell. By "outer layer" is meant in the context of the present invention the shell layer furthest from the nanocrystal core and in direct contact with the medium or the environment in which the nanocrystal is located. A multilayer shell may comprise from 2 to 10, in particular from 2 to 5 different semiconductor layers. Thus, different variants are possible for the shell of the nanocrystal prepared by the method of the invention.

In a first variant, x in the formula ZnSi_ _x F _x is equal to 0 and the shell is formed only of a layer which is therefore a layer of ZnS.

In a second variant, x in the formula ZnSi_ _x F _x is such that 0 <x <1 and the shell is formed of only one layer. In a third variant, x in the formula ZnSi_ _x F _x is equal to 0 and the shell comprises at minus two different layers among which the outer layer is a layer of ZnS.

In a fourth variant, x in the formula ZnSi_ _x F _x is such that 0 <x <1 and the shell comprises at least two different layers.

In addition, the layer (s) of the shell of the nanocrystal prepared according to the method (2) of the invention can (wind) have a uniform chemical composition or, inside, a same layer, a chemical composition which differs and in particular a chemical composition in the form of a gradient. In this case, the outer part of the shell will be constituted by the outer zone of a shell with a layer and by the outer zone of the outer layer of a multilayer shell.

When the shell is multilayer, the layer (or layers) included between the core of the nanocrystal and the outer layer of formula ZnSi _× F _x as defined above may comprise a semiconductor of ternary composition (A, B, C), and, more particularly, of formula ABC2, as defined above, and / or a semiconductor of formula DE as defined above.

The metal or metalloid D in the +11 oxidation state is chosen in particular from magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), zinc (Zn), cadmium (Cd), mercury

(Hg), tin (Sn), lead (Pb) and mixtures thereof.

The element E in the oxidation state -II is in particular chosen from oxygen (O), sulfur (S), selenium (Se), tellurium (Te) and mixtures thereof. Examples semiconductors that may be present in the layers between the core of the nanocrystal and the outer layer of the shell are, for example, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnO, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, SnS, SnSe, SnTe, PbS, PbSe, PbTe and mixtures thereof. More particularly, examples of semiconductors that may be present in the layers between the core of the nanocrystal and the outer layer of the shell are selected from the group consisting of MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe , BaS, BaSe, BaTe, ZnO, ZnS, ZnSe, ZnTe, SnS, SnSe, SnTe and mixtures thereof.

It is therefore clear that, in the case of a multilayer shell, the reaction mixture used in the context of the present invention and in particular in step (cb) also contains the precursors of the elements constituting the layers other than the outer layer of the shell.

Step (cb) allows, on the one hand, to add, to the mixture obtained in step (c), ie to the mixture containing nanocrystals of type (A, B, C), and in particular ABC2, forming the core nanocrystals prepared by implementation of the method (2), the precursors of the (or) shell (s) surrounding or coating such a core and, secondly, to maintain the mixture at the temperature T _c at which the shell is formed. All the precursors used in the process (2) of the present invention are either commercially available products, ie products for which the person skilled in the art knows at least one preparation process. Thus, at least one of the precursors of zinc, sulfur and F may have been optionally prepared beforehand before introduction into the mixture of step (cb) or be prepared in situ in said mixture.

In the context of the present invention, the zinc precursor used is chosen from the group consisting of zinc salts, zinc halides, zinc oxides and organometallic zinc compounds. By "organometallic compound of zinc" is meant, more particularly, a bi-substituted zinc compound, a zinc carboxylate or a zinc phosphonate.

By "bi-substituted zinc compound" is meant in the context of the present invention a compound of formula (R ₁ ) 2 Zn in which each R ₄ , identical or different, represents a hydrocarbon group of 1 to 20 carbon atoms such an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical.

By "zinc carboxylate" is meant in the context of the present invention a compound of formula (R ₁ COO) 2Zn in which each R ₅ , identical or different, represents a hydrocarbon group of 1 to 20 carbon atoms such as an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical. A carboxylate advantageously used is zinc stearate. By "zinc phosphonate" is meant in the context of the present invention a compound of formula [R ₁ -P (OR 1 ₇ ) (OR 1 ₈ ) O] ₂ Zn in which: each R ₆ , identical or different, represents a hydrocarbon group of 1 to 20 carbon atoms such as an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical; each R ₁₇ , identical or different, represents a hydrogen atom, a hydrocarbon group of 1 to 20 carbon atoms such as an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical, and each Ris, identical or different, represents a hydrogen atom, a hydrocarbon group of 1 to 20 carbon atoms such as an alkyl radical, an alkenyl radical, an alkoxy radical, an aryl radical or an aryloxy radical.

The alkyl, alkenyl, alkoxy, aryl and aryloxy radicals are as defined for the precursors of A.

In the context of the present invention, the sulfur precursor used is chosen from the group consisting of an aliphatic thiol, elemental sulfur dissolved in an organic solvent, a xanthate and a compound of formula S (Si (1 * 19) 3) 2 in which each R 19, identical or different, is a linear, branched or cyclic, optionally substituted, alkyl group of 1 to 10 carbon atoms, in particular from 1 to 6 carbon atoms and, in particular, from 1 to 3 carbon atoms.

The sulfur precursors such as the aliphatic thiol and the xanthates, and the forms of implementation of these as the organic solvent used are as previously defined.

In the context of the present invention, the precursor of F possibly used in step (cb) is chosen from the group consisting of an oxygen precursor, a selenium precursor, a tellurium precursor and their mixtures.

Advantageously, the precursor of F is selected from elemental selenium dissolved in an organic solvent; elemental tellurium dissolved in an organic solvent; zinc acetate; a phosphine selenide; a phosphine oxide; a compound of formula F '(Si (1 * 20) 3) 2 in which F' represents Se or Te and each R20, which is identical or different, is a linear, branched or cyclic alkyl group, optionally substituted, of 1 to 10 atoms carbon, especially from 1 to 6 carbon atoms and, in particular, from 1 to 3 carbon atoms, and mixtures thereof. The precursors of F such as phosphines selenide and oxide, and the forms of implementation thereof as the organic solvent used are as previously defined.

The concentration of the zinc precursor and that of the sulfur precursor, optionally with the precursor of F is between 5 and 400 mmol / L, especially between 10 and 300 mmol / L and in particular between 20 and 200 mmol / L.

When the shell of the nanocrystal is multilayer, the precursors of the various elements, metals and metalloids constituting the layers other than the outer layer of the shell, are present in the reaction mixture at a concentration of between 5 and 400 mmol / L, especially between 10 and 300 mmol / L and in particular between 20 and 200 mmol / L.

The addition of the precursors of zinc, sulfur and possibly F and other metals, metalloids or elements constituting the inner layers of the shell during step (cb) can be done in different ways.

In a first variant, the precursors to be added are premixed together and the mixture thus obtained is added at once to the mixture of step (c).

In a second variant, the precursors to be added are premixed together and the mixture thus obtained is added at least twice to the mixture of step (c). In this variant, the precursor mixture is advantageously added in the form of a drop by drop.

In a third variant, the precursors to be added are added independently of each other to the mixture of step (c), each precursor being able to be added to said mixture in one alone or at least twice, or even in the form of a drip.

In a fourth variant, certain precursors are mixed together and the mixture (or mixtures) thus obtained is (are) added (s) to the mixture of step (c) in a single or at least two time, even in the form of a drip, while at least one other precursor is added, independently of this (or these) mixture (s), to the mixture of step (c) and this, in one or at least twice, or even in the form of a drip.

Whatever the variant used for the addition of the precursors, they can be added in solid form, especially in powder form or in the form of a solution. When at least one precursor of the precursors of zinc, sulfur and, optionally, F and other metals, metalloids or elements constituting the inner layers of the shell is added in solution, this solution comprises, as solvent, a solvent organic as previously defined.

The temperature of the mixture during step (cb) is maintained at the temperature T _c as previously defined. It should be noted that it may be necessary to bring the temperature of the mixture of step (c) to the temperature T _c , if the temperature of said mixture decreases between steps (c) and (cb). The different means and forms of implementation to bring and / or maintain the mixture to the temperature T _c during step (cb) are identical to those envisaged for steps (b) and (c).

Step (cb) advantageously has a duration of between 5 min and 5 h, in particular between 10 min and 3.5 h and, in particular, between 20 min and 2 h. Advantageously, the duration of step (cb) is of the order of 30 min. By "duration of the order of 30 min" is meant a duration of 30 min ± 10 min and especially a duration of 30 min ± 5 min. The precursors of zinc, sulfur, F and other metals, metalloids or elements constituting the inner layers of the shell may be added to the mixture throughout the duration of step (cb) or have been added in the first minutes of step (cb). By "the first minutes" is meant within the scope of the present invention the l ^st, the 2 1 ^st, the 5 1 ^st, 1 ^st 15 minutes of step (cb).

The purification step (θ2) of the process (2) is a purification / separation step of the core / shell nanocrystals thus prepared, to which the embodiments and variants previously envisaged for step (di) apply. .

The present invention relates to any nanocrystal obtainable by a method according to the present invention, i.e.

or a nanocrystal comprising a semiconductor of ternary composition (A, B, C), and more particularly of formula ABC2, or a nanocrystal having (i) a core comprising a semiconductor of ternary composition (A, B, C), and, more particularly, of formula ABC2, with A representing a metal or metalloid in the +1 oxidation state B being a metal or metalloid in oxidation state + III and C representing an element in the oxidation state -II, coated with (ii) a shell whose external part comprises a semiconductor of formula ZnSi_ _x F _x , with F representing an element in the oxidation state -II and x being a decimal number such that 0 ≤ x <1.

More particularly, the present invention relates to a nanocrystal having a core comprising a semiconductor comprising copper, indium and sulfur, coated by a shell whose outer portion comprises a semiconductor comprising zinc and sulfur, capable of to be obtained by a process according to the invention.

Indeed, the use of a semiconductor material comprising zinc and sulfur in the outer part of the shell has several advantages:

1) this material is non-toxic and has good chemical stability;

2) its mesh parameter (5.41 A) is close to that of the CIS core (5.52 A), ie a mismatch of about 2%, a weak mismatch between the core and shell materials being essential in order to to be able to grow the shell epitaxially and thus avoid the formation of crystalline defects that may reduce the fluorescence efficiency; 3) its wide gap band gap of 3.8 eV ("band gap" in English) and its alignment of energy bands relative to the CIS ensure the confinement in the CIS charge carriers generated during a light excitation. As a result, the ZnS shell does not influence (or very little) the emission wavelength. On the other hand, the fluorescence intensity and photo-stability can be greatly improved thanks to the better passivation of the CIS surface and its physical separation from the surrounding environment by the ZnS shell.

More particularly, the nanocrystal object of the present invention, ie having a core comprising a semiconductor comprising copper, indium and sulfur, coated by a shell whose outer portion comprises a semiconductor comprising zinc and sulfur, obtainable by a process according to the invention has a fluorescence quantum yield higher than the values reported to date. The fluorescence quantum yield with the nanocrystals according to the invention is greater than 5% at ambient temperature, especially greater than 10% at room temperature, in particular greater than 20% at ambient temperature and, more particularly, greater than 50%.

Those skilled in the art know different techniques for obtaining, for a given nanocrystal, its fluorescence quantum yield at room temperature. As an example, one can cite the technique of comparing the intensity spectrally integrated emission of a nanocrystal dispersion according to the invention in hexane of optical density X at an excitation wavelength Y with that of a solution of rhodamine G6 in ethanol of same optical density and at the same excitation wavelength.

The nanocrystal having a core comprising a semiconductor comprising copper, indium and sulfur, coated by a shell whose outer part comprises a semiconductor comprising zinc and sulfur, object of the present invention emits in the near infrared, advantageously in the spectral range of 500 to 900 nm and in particular in the spectral range of 650 to 900 nm, which is particularly interesting for in vivo optical imaging.

The present invention also relates to a composition comprising at least one nanocrystal having a core comprising a semiconductor comprising copper, indium and sulfur, coated by a shell whose external part comprises a semiconductor comprising zinc and aluminum. sulfur in an aqueous medium. Said composition is advantageously a liquid composition.

By "aqueous medium" is meant in the context of the present invention a medium selected from the group consisting of water, demineralised water, deionized water, a saline solution such as PBS, a physiological solution, solution of sodium chloride, aqueous acetic acid, a mixture of water and an organic solvent as defined above and mixtures thereof.

Indeed, once synthesized, the nanocrystals according to the present invention are in an organic medium and need to be transferred to an aqueous medium before any use in biology and in particular for in vivo molecular imaging.

The passage of nanocrystals in an aqueous medium is generally carried out by any technique known to those skilled in the art. These techniques involve (i) an exchange of ligands (ie the initial ligands stabilizing the nanocrystals in an organic medium by ligands which will stabilize the nanoparticles in an aqueous medium), (ii) the formation of an intermediate layer between the nanocrystal and the nanocrystal. aqueous medium such as a layer of silica, or

(iii) the hydrophobic / hydrophobic interaction with at least one amphiphilic polymer with the hydrophobic portion of the polymer interacting with the initial organic ligands stabilizing the nanocrystals, and the hydrophilic portion for stabilizing said nanocrystals in aqueous buffer. For all these techniques, we talk about functionalization of nanocrystals.

Therefore, the method (2) according to the present invention may have an additional step for functionalizing the purified nanocrystals after step (θ2).

More particularly, in the context of the ligand exchange, the passage of the nanocrystals in aqueous buffer is carried out under conditions which favor a maximum exchange rate of ligands such as than dodecanethiol by the new amphiphilic ligands and a stability of the nanocrystals thus functionalised in aqueous suspension. Any amphiphilic ligand known to those skilled in the art can be used in this functionalization. By way of examples, mention may be made of amphiphilic ligands of the mercaptocarboxylic acid type, of the β-diketone type and dihydrolipoic acid. The latter has a small size, a group charged to promote solubilization in water, and two thiol functions known for their high affinity with the surface of nanocrystals comprising a shell of zinc sulfide.

The present invention finally relates to the use of a nanocrystal capable of being prepared according to a method of the invention or a nanocrystal according to the invention in a light-emitting diode or in a photovoltaic cell.

The present invention finally relates to the use of a nanocrystal capable of being prepared according to a process of the invention, a nanocrystal according to the invention or a composition according to the present invention for the fluorescent marking of chemical molecules or organic.

To summarize the particular advantages provided by the present invention, the interest of the proposed method is multiple:

1. It gives nanocrystals based on CIS / ZnS having a higher fluorescence quantum yield than the nanocrystals known hitherto and in particular greater than 20% at ambient temperature in the visible spectrum (480-650 nm) and greater than 5% in the near infrared (650-900 nm).

2. It gives access to CIS / ZnS nanocrystals emitting in the near infrared and in particular in the 650-900 nm spectral range, which is particularly interesting for in vivo optical imaging.

3. It is very simple, which facilitates the change of scale in order to increase the quantity produced. In particular, no pyrophoric precursor is used and the synthesis steps of the heart and core / shell crystals follow each other directly, i.e. without intermediate purification of the CIS core crystals. In addition, no sorting step in size (e.g. by selective precipitation) is required because of the small size dispersion of the samples obtained directly after the synthesis.

4. After proper functionalisation of the surface, the CIS / ZnS nanocrystals can be transferred to an aqueous medium and used as a fluorescent marker in biology. They do not contain cadmium, lead or mercury, heavy metals with acute or chronic toxicity. Their emission properties are sufficiently powerful to allow the detection of their biodistribution in vivo without sacrificing the animal in Nude mice at doses of the order of 10 ¹⁷ copper atoms / mouse of 20 g.

Other features and advantages of the present invention will become apparent to those skilled in the art upon reading the examples below given to illustrative and non-limiting, and with reference to the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the UV-vis absorption spectra of the samples taken during the experiment described in Part II below (synthesis of CIS nanocrystals), using a reaction temperature of 200 ^° C. Figure 2 shows the photoluminescence spectra of the samples taken during the experiment described in Part II below (synthesis of CIS nanocrystals). The excitation wavelength is 470 nm. FIG. 3 shows the photoluminescence spectra of the CIS / ZnS samples prepared according to part III below, using for the synthesis of CIS core nanocrystals a temperature of 230 ^° C. and a reaction time of 20 min (FIG. 3A); 40 min (Figure 3B); 60 min (Figure 3C). The excitation wavelength is 470 nm.

FIG. 4 shows an X-ray powder diffractogram of the CIS core nanocrystals prepared by heating at 270 ^° C. for 30 min (Curve A), CIS core nanocrystals prepared by heating at 230 ^° C. for 40 min (Curve B), and CIS / ZnS core / shell nanocrystals (Curve C), made from sample B.

Figure 5 shows transmission electron microscopy images of a sample of CIS core nanocrystals prepared in heating at 230 ⁰ C for 40 min (Figure 5A) and sample heart / shell CIS / ZnS corresponding to the same magnification (Figure 5B).

FIG. 6 shows the photoluminescence spectra of the CIS-ZnS nanocrystals before and after transfer in IX PBS aqueous buffer (excitation at 590 nm).

Figure 7 shows the fluorescence images showing the bio-distribution over time of nanocrystals CIS-ZnS (6, 5.10 ¹⁶ - l, 3.10 ¹⁷ copper atoms) injected intravenously.

(caudal vein) in healthy Nude mice.

DETAILED PRESENTATION OF PARTICULAR EMBODIMENTS

All manipulations of air-sensitive materials are done in a glove box or using a vacuum ramp (Schlenk technique).

I. Material and methods . For the characterization, the UV-visible absorption spectra were measured on a Hewlett-Packard 8452A spectrometer (wavelength spectral domain: 190-820 nm, resolution 2 nm), the photoluminescence spectra were acquired with Hitachi F-4500 spectrometer. For these spectroscopic measurements, dilute colloidal solutions of nanocrystals in hexane were placed in 1 cm optical path quartz cuvettes. The fluorescence quantum yields at room temperature were obtained by comparing the emission intensity - spectrally integrated - of the solution of nanocrystals in hexane with that of a 6G rhodamine solution in ethanol, both solutions having the same optical density (<0.03) at the excitation wavelength (490 nm). RX diffractograms were obtained on a Philips X'Pert device, using a Co source at 50 kV and 35 mA. Transmission electron microscopy images were acquired with a JEOL 4000FX microscope.

All products except zinc stearate (Riedel de Haën) and copper iodide (Acros) were purchased from Sigma-Aldrich and used as such, indium acetate (purity 99.99%), copper iodide ( purity 99.995%), zinc stearate (purity 90%), dodecanethiol (purity 97%) and 1-octadecene (purity 90%). The synthesis of zinc ethylxanthate used in shell growth is described below: 0.005 mol of cadmium chloride is dissolved in 20 ml of distilled water. A solution of 0.01 mol of potassium ethylxanthogenate dissolved in 20 ml of distilled water is added to it. A white precipitate is thus formed, which is filtered and then washed 3 times with 50 ml of distilled water and dried under vacuum.

II. Synthesis of CIS nanocrystals. II. A. Protocol.

Step 1: 0.3 mmol of copper (I) iodide, 0.3 mmol of indium (III) acetate, 12.5 mmol of 1-dodecanethiol and 25 ml of 1-octadecene are placed in a flask 50 mL tricol equipped with a condenser and mixed under a stream of inert gas (argon or nitrogen) using a magnetic stirrer. Step 2: This mixture is heated at 50 ^° C. for 1 h under a primary vacuum and then purged with nitrogen or argon.

Step 3: The reaction mixture is heated at 230 ^° C., and it is left stirring at this temperature for 40 min. For the rise in temperature, during which the reaction mixture becomes transparent, a ramp of about 150 ° C / minute is used. Step 4: After cooling to room temperature, the CIS nanocrystals can be isolated by adding a volume equivalent of a chloroform / methanol mixture (1: 1 vol: vol) and 10 equivalents by volume of acetone, then by centrifugation. The resulting precipitate containing the nanocrystals can be dispersed in organic solvents such as hexane, toluene or chloroform.

II. B. Characterization of CIS nanocrystals.

The UV-vis absorption and photoluminescence absorption spectra of the CIS nanocrystals before coating with a ZnS shell, obtained during a synthesis carried out at 200 ^° C., are shown in FIGS. 1 and 2, respectively.

In Figure 1, we observe the shift of the absorption threshold to longer wavelengths with time, indicating the increase in the size of the nanocrystals during the reaction. From 130 min, this evolution stagnates. In the photoluminescence spectra

(Figure 2), there is a signal having a maximum at around 650 nm to 20 min. The peak moves with the reaction time to longer wavelengths, which is accompanied by the appearance of two peaks located at about 713 and 806 nm. The relative intensity of these three signals also varies over time. The photoluminescence spectra of CIS generally have several lines that come from transitions involving electronic states inside the gap (forbidden band of the semiconductor). These are close to the valence band and close to the conduction band, giving rise to donor-acceptor transitions, for example. The observed maximum fluorescence quantum yield of the CIS core crystals prepared according to the method of the present invention is of the order of 8%.

III. Synthesis of core / shell nanocrystals

CIS / ZnS.

III. A. Protocol.

For the growth of the shell in ZnS, the reaction described in part II is continued after step 3, that is to say before purification.

The temperature of the reaction mixture is maintained (or adjusted in the case where the synthesis of CIS crystals has been carried out at another temperature) at

230 ⁰ C and the precursors of ZnS, a mixture of 2.5 mmol of zinc stearate and 0.32 mmol of ethylxanthate of zinc in 20 mL of 1-octadecene are added dropwise over 30 min.

After cooling to room temperature, the purification of the CIS / ZnS nanocrystals is carried out in the same manner as described for CIS crystals, i.e. step 4 of Part II.

III. B. Characterization of CIS / Zns core / shell nanocrystals and comparison with CIS nanocrystals.

Figure 3 shows the evolution of the photoluminescence spectra during the growth of the ZnS shell on three different samples of CIS nanocrystals. In Figure 3A, both a more symmetrical shape of the fluorescence line, a narrowing of its width from 120 nm (FWHM, width at half-height) to 100 nm and an increase in integrated intensity (500 -900 nm) by a factor> 7. The quantum yield of this sample was determined at 61%.

The photoluminescence spectrum of the CIS core sample visible in FIG. 3B has two distinct peaks at 723 and 802 nm, as well as a shoulder at around 680 nm. During the coating with the ZnS shell, the peaks at 682 and 723 nm, whose intensity increases most strongly. The integrated intensity increases by a factor of 5 and the fluorescence quantum yield of the sample is 42%. Figure 3C shows that it is also possible to specifically increase the intensity of the line with longer wavelength (810-820 nm). An improvement in integrated intensity is observed at a factor of 4.1 giving a quantum yield of 8%. These results show that a wide spectral range can be covered with CIS / ZnS nanocrystals. Coating with ZnS makes it possible to significantly increase the fluorescence quantum yield.

In Figure 4, powder X-ray diffractograms, obtained for heart and heart / shell samples, are compared. The diffractograms of samples A and B show the characteristic peaks of the cubic phase of CuInS2 (map of Joint Committee on Powder Diffraction Standards, JCPDS, No. 001-8517). These two samples contain CIS nanocrystals of different sizes. The larger peak width for sample B indicates that the size of the crystallites is smaller. The diffractogram of the C sample confirms the formation of the CIS / ZnS heterostructure. Peaks include both the contribution of the CIS cubic phase and the ZnS zinc blende structure (JCPDS map No. 05-0566).

Figure 5 compares transmission electron microscopy images of the CIS nanocrystals before and after growth of the shell into ZnS. There is an increase in the average size of 3 nm to 7 nm corresponding to the deposition of a shell consisting of about 6 monolayers of ZnS. The size distribution of the core / shell sample is less than 10%. III. C. Functionalization of CIS / ZnS nanocrystals.

All products except NAP-5 (GE Healthcare) columns were purchased from Sigma-Aldrich. Firstly, in order to eliminate the excess of dodecanethiol used in the synthesis step of the nanocrystals, the nanocrystals are washed twice by the addition of a non-solvent (ethanol) in the following manner. A solution of 250 μl of hexane containing the CIS-ZnS nanocrystals (synthesized as described above) is placed in a 1.5 ml microtube. 250 μL of ethanol are added to precipitate the nanocrystals. The microtube is centrifuged at 13,000 x g for 10 min on a Thermo Electro Corporation model HERAEUS PICO17 centrifuge. The CIS-ZnS nanocrystals are separated from the supernatant and then redispersed in 250 μl of hexane.

After the washing step, the nanocrystals are dried under vacuum at 50 ^° C. Once the residue of hexane and ethanol is evaporated, 40 μl of dihydrolipoic acid are added and the reaction mixture is heated to 70 ^° C. magnetic stirring. After two hours of heating, 1 mL of dimethylformamide is added to solubilize the contents. Then an excess of potassium tert-butoxide is added, resulting in the precipitation of the nanocrystals. The microtube is centrifuged at 13,000 xg for 10 min on a Thermo Electro Corporation model HERAEUS PICO17 centrifuge. The CIS nanocrystals are separated from the supernatant and redispersed in a buffer solution (0.01 M sodium phosphate and 0.15 M sodium chloride) with a pH of 7.4.

Finally, this solution of CIS-ZnS nanocrystals is purified on a Sephadex G25 NAP-5 column (GE Healthcare) to eliminate the excess of potassium tert-butoxide. A small amount of dihydrolipoic acid is added to the purified solution to increase the colloidal stability of the product.

IV. Intravenous injection of functionalized CIS / ZnS nanocrystals.

Once ligand exchange has been achieved, three conditions must be met in order to use the product for biological applications, including injecting it into the small animal for in vivo fluorescence imaging. These conditions are as follows:

knowledge of the concentration of the solutions: the different elements constituting the nanocrystal present in the solution are assayed by an inductively coupled plasma mass spectrometry (ICP-MS) analysis after treatment with 65% nitric acid; the absence of agglomerates: the hydrodynamic diameter of the nanoparticles obtained in aqueous PBS IX buffer after ligand exchange is determined using the NANO-ZS nanosizer from Malvern: a hydrodynamic diameter of 17 ± 3 nm is obtained; a high optical signal to have good detection sensitivity. The optical properties of these nanocrystals are determined after the passage in the aqueous phase. Figure 6 shows the photoluminescence spectra obtained before and after passage in aqueous buffer (excitation at 590 nm). A red shift of approximately 50 nm from the emission wavelength of the nanocrystals is observed after transfer into aqueous buffer.

Once these conditions have been verified, 200 μl of the aqueous solution of functionalized CIS-ZnS nanocrystals corresponding to 6, 5.10 ¹⁶ - 1, 3 × ^{10 17} copper atoms, are injected intravenously into the tail of female Nude mice aged from 6 to 8. weeks, and maintained under conditions without pathogens (IFFA-Credo, Marcy l'Etoile, France).

The mice were maintained under gaseous general anesthesia (isoflurane) throughout the experiment. The anesthetized mice were imaged with a Reflectance Fluorescence Imaging (FRI) device comprising as a source of excitation a ring of LEDs provided with interference filters, emitting at 633 nm (power of illumination 50 μW.cm ^"2 ) as described for example in the article by Texier et al., 2005 [9].

The images were collected after filtration by an optical density> 5 RG665 color filter at the excitation wavelength by a CCD camera (Orca BTL, Hamamatsu) with an exposure time of 500 ms. The signals were quantified using the Wasabi image processing software. Figure 7 shows the evolution as a function of time of the fluorescence of nanocrystals CIS-ZnS functionalized with dihydrolipoic acid in the mouse over a period of 24 hours. No change in the behavior of the animal is observed during the 24 hours following the injection and no sign of toxicity is detected.

Nanocrystals are directed to the liver and lungs as early as 15 minutes after injection. Fecal elimination is observed 6 h after injection. These non-invasive observations in vivo are confirmed by fluorescence organ analysis after sacrifice of the animal at 24 h.

In conclusion, the CIS-ZnS nanocrystals functionalized with dihydrolipoic acid are suitable for in vivo fluorescence imaging. Their subsequent functionalization by different targeting ligands (anti-bodies, peptides, saccharides) making it possible to direct them towards other areas of interest to be imaged can be envisaged by grafting these so-called targeting ligands on the acid function of dihydrolipoic acid. . In addition, at least partial elimination of these nanocrystals by the faecal route should allow their repeated use in imaging protocols, especially in order to evaluate the effectiveness of therapeutic treatments.

REFERENCES

[1] H. S. Choi, W. Liu, P. Misra, E. Tanaka, J. P. Zimmer, B. I. Ipe, M. G. Bawendi, J. V. Frangioni, Nat. Biotechnol. 2007, 25, 1165-1170.

[2] J. P. Zimmer, S. W. Kim, S. Ohnishi, E. Tanaka, J. V. Frangioni, M. G. Bawendi, J. Am. Chem. Soc. 2006, 128, 2526-2527.

[3] S. L. Castro, S. Bailey, R. Raffaelle, K. K. Banger, A. F. Hepp, Chem. Mater. 2003, 15, 3142-3147.

[4] S. L. Castro, S. Bailey, R. P.

Raffaelle, K.K. Banger, A.F. Hepp, J. Phys. Chem. B 2004, 108, 12429-12435.

[5] J. J. Nairn, P. J. Shapiro, B. Twamley, T. Pounds, R. von Wandruszka, T. R. Fletcher, M.

Williams, C.M. Wang, G. Norton, Nano Lett. 2006, 6, 1218-1223.

[6] K. K. Banger, M.H.C. Jin, J.D. Harris, P.E. Fanwick, A.F. Hepp, Inorganic Chemistry 2003, 42, 7713-7715.

[7] H. Nakamura, W. Kato, M. Uehara, K. Nose, T. Omata, S. Otsuka-Yao-Matsuo, M. Miyazaki, H. Maeda, Chem. Mater. 2006, 18, 3330-3335. [8] Pan D., L. An, Sun Z., W. Hou, Y. Yang, Z. Yang, Y. Lu, J. Am. Chem. Soc. 2008, 130, 5620-5621.

[9] Texier, I. et al., Proceedings of the SPIE 2005, 5704, 16-22.

Claims

1) Process for the preparation of a nanocrystal comprising a ternary semiconductor compound consisting of elements A, B and C with A representing a metal or metalloid in the oxidation state +1, B representing a metal or metalloid at the oxidation state + III and C representing an element in oxidation state -II, characterized in that said process comprises the successive steps of: a) preparing a mixture comprising at least one precursor of A, at least one precursor of B and at least one precursor of C at a temperature T _a ; b) maintaining the mixture obtained in step (a) at a temperature T _b greater than or equal to the temperature T _a ; c) bringing the mixture obtained in step (b) of the temperature T _b to a temperature T _c greater than the temperature T _b ; di) optionally purifying the nanocrystals comprising a ternary semiconductor compound consisting of elements A, B and C obtained in step (c). 2) Process for the preparation of a nanocrystal having (i) a core comprising a ternary semiconductor compound consisting of elements A, B and C with A representing a metal or metalloid in the oxidation state +1, B representing a metal or metalloid in the oxidation state + III and C representing an element in the oxidation state -II, coated by (ii) a shell whose external part comprises a semiconductor of formula ZnSi_ _x F _x , with F representing an element in oxidation state -II and x being a decimal number such that 0 ≤ x <1, characterized in that said method comprises the steps of: α) preparing a nanocrystal comprising a semiconducting ternary compound consisting of elements A, B and C, according to a process as defined in claim 1, β) coating the nanocrystal prepared in step (α) with a shell whose outer part comprises a semiconductor of formula ZnSi_ _x F _x , with F representing an element in the oxidation state -II and x being a decimal number such that 0 ≤ x <1. 3) Process according to claim 2, characterized in that said process comprises the successive steps of: a) preparing a mixture comprising at least one precursor of A, at least one precursor of B and at least one precursor of C at a temperature T _a ; b) maintaining the mixture obtained in step (a) at a temperature T _b greater than or equal to the temperature T _a ; c) bringing the mixture obtained in step (b) of the temperature T _b to a temperature T _c greater than the temperature T _b ; cb) adding, to the mixture obtained in step (c) and maintained at temperature T _c , at least one zinc precursor, at least one sulfur precursor and optionally at least one precursor of F; θ2) purifying the nanocrystals having a core comprising a ternary semiconductor compound consisting of elements A, B and C, coated with a shell whose outer layer comprises a semiconductor of formula ZnSi _× F _x , obtained in step (cb). 4) Process according to any one of claims 1 to 3, characterized in that said ternary compound is of formula ABC2. 5) Process according to any one of the preceding claims, characterized in that said precursor of A is selected from the group consisting of a copper precursor, a silver precursor, and mixtures thereof. 6) Process according to any one of the preceding claims, characterized in that said precursor of A is chosen from the salts of A, the halides of A, the oxides of A and the organometallic compounds of A. 7) Process according to any one of the preceding claims, characterized in that said precursor of B is selected from the group consisting of an indium precursor, a gallium precursor, an aluminum precursor and mixtures thereof. 8) Process according to any one of the preceding claims, characterized in that the said precursor of B is chosen from the salts of B, the B halides, B oxides and organometallic compounds of B. 9) Process according to any one of the preceding claims, characterized in that said precursor of C is selected from the group consisting of a sulfur precursor, an oxygen precursor, a selenium precursor, a tellurium precursor and mixtures thereof . 10) Process according to any one of the preceding claims, characterized in that said precursor of C is selected from elemental selenium dissolved in an organic solvent; elemental tellurium dissolved in an organic solvent; elemental sulfur dissolved in an organic solvent; an aliphatic thiol; a xanthate; an amine oxide; a phosphine selenide; a phosphine oxide; a compound of formula C (Si (Rn) 3) 2 in which C represents a member selected from the group consisting of S, Se and Te and each Rn, which is the same or different, is a linear, branched or cyclic alkyl group, optionally substituted , from 1 to 10 carbon atoms. 11) Process according to any one of the preceding claims, characterized in that said mixture prepared in step (a) is carried out in an organic solvent. 12) Process according to any one of the preceding claims, characterized in that said mixture prepared in step (a) contains a member selected from the group consisting of a stabilizer for the surface of the nanocrystals and a primary amine. 13) Process according to any one of the preceding claims, characterized in that said temperature T _a during step (a) is less than 50 ⁰ C, especially less than 40 ⁰ C and, in particular, less than 30 ⁰ vs. 14) Method according to any one of the preceding claims, characterized in that said temperature T _b during step (b) is less than 100 ⁰ C, especially between 30 and 80 ⁰ C, in particular between 40 and 60 ^° C. 15) Process according to any one of the preceding claims, characterized in that said temperature T _c is greater than 150 ⁰ C, in particular greater than 180 ⁰ C, in particular between 180 ⁰ C and 300 ⁰ C, and more particularly between 200 ° C and 270 ° C. 16) Process according to any one of claims 3 to 15, characterized in that said zinc precursor is selected from the group consisting of zinc salts, zinc halides, zinc oxides and organometallic zinc compounds. 17) Process according to any one of claims 3 to 16, characterized in that said precursor of F is selected from the group consisting of an oxygen precursor, a selenium precursor, a tellurium precursor and mixtures thereof. 18) Process according to any one of claims 3 to 17, characterized in that said step (cb) has a duration of between 5 min and 5 h, in particular between 10 min and 3.5 h and, in particular, between 20 min. min and 2 h. 19) Nanocrystal having a core comprising a semiconductor comprising copper, indium and sulfur, coated by a shell whose outer portion comprises a semiconductor comprising zinc and sulfur, obtainable by a Method according to any one of Claims 2 to 18, characterized in that the said nanocrystal has a quantum yield greater than 5% at ambient temperature, in particular greater than 10% at ambient temperature, in particular greater than 20% at ambient temperature, and more particularly, greater than 50%. 20) Nanocrystal according to claim 19, characterized in that it emits in the spectral range of 500 to 900 nm and in particular in the spectral range of 650 to 900 nm. 21) Composition comprising at least one nanocrystal according to any one of claims 19 or 20 in an aqueous medium. 22) Use of a nanocrystal according to any one of claims 19 or 20 in a light emitting diode or in a photovoltaic cell. 23) Use of a nanocrystal according to any one of claims 19 or 20 or a composition according to claim 21 for the fluorescent labeling of chemical or biological molecules.