Classifications

• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
  • C09K11/00—Luminescent materials, e.g. electroluminescent or chemiluminescent
  • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
  • C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
  • A61K49/0065—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle
  • A61K49/0067—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle quantum dots, fluorescent nanocrystals
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
  • C09K11/00—Luminescent materials, e.g. electroluminescent or chemiluminescent
  • C09K11/08—Luminescent materials, e.g. electroluminescent or chemiluminescent containing inorganic luminescent materials
  • C09K11/56—Luminescent materials, e.g. electroluminescent or chemiluminescent containing inorganic luminescent materials containing sulfur
  • C09K11/562—Chalcogenides
  • C09K11/565—Chalcogenides with zinc cadmium
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
  • C09K11/00—Luminescent materials, e.g. electroluminescent or chemiluminescent
  • C09K11/08—Luminescent materials, e.g. electroluminescent or chemiluminescent containing inorganic luminescent materials
  • C09K11/88—Luminescent materials, e.g. electroluminescent or chemiluminescent containing inorganic luminescent materials containing selenium, tellurium or unspecified chalcogen elements
  • C09K11/881—Chalcogenides
  • C09K11/883—Chalcogenides with zinc or cadmium
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y40/00—Manufacture or treatment of nanostructures

Definitions

• the present invention refers to the field of luminescent semiconductor nanocrystals (quantum dots).
• the present invention relates to the functionalization of quantum dots (QD) surface by exchanging the native ligands with other ligands capable of adjusting the solubility of said nanoparticle, in particular in water and other polar solvents.
• QD quantum dots
• Quantum dots are semiconductor nanocrystals endowed with unique optical and electronic properties, such that they are emerging as substitutes for molecular fluorophores in a variety of technological applications.
• QDs of, e.g., CdSe exhibit a high light absorption and an intense luminescence in the UV-visible region, whose wavelength can in both cases be modulated by adjusting the diameter of the particle.
• QDs are chemically and photochemically very stable, and are excellent two-photon absorbers. For these peculiar properties, QDs are employed in several applications related to the use of luminescence: (bio)chemical analysis, diagnostic imaging, medical therapy, photovoltaic solar cells, and LED devices for lighting and displays.
• Another reason for modifying the capping layer of QDs is to link functional molecular units (e.g., receptors, fluorophores, switches, sensitizers, biomolecules) to the nanocrystal, with the aim of developing hybrid nanomaterials with predetermined properties.
• functional molecular units e.g., receptors, fluorophores, switches, sensitizers, biomolecules
• the surface can be made water-compatible following two main strategies: 1 ) exchange of the native ligands with capping agents that combine an anchoring group for the metal surface and a hydrophilic part; 2) encapsulation of the hydrophilic nanocrystals using amphiphilic molecules or polymers.
• the first strategy allows for the preparation of hydrophilic nanocrystals with a compact shell layer (i.e. with small overall size, suitable for crossing biological barriers) but with reduced luminescence quantum yield (because of the non-optimal surface passivation obtained after ligand exchange).
• the surface ligands should have two main functional domains: a hydrophilic moiety that allows the solubilization of nanocrystals in polar media, and an anchoring group which can bind the surface of the quantum dot.
• Thiols are widely employed anchoring groups. Examples of the first strategy include the substitution of the hydrophobic ligand with carboxy-terminated thiols, such as mercaptopropionic and mercaptoacetic acid and thiol-containing zwitterionic molecules, such as cysteine.
• the surface functionalization using monothiolated ligands while it is a simple approach, affords nanocrystals with lower emission quantum yield (this is true in general for all the hydrophilic nanocrystals obtained following the first strategy) and poor long-term stability, due to the desorption of the ligands from the surface.
• Bis-thiol derivatives such as dihydrolipoic acid, dihydrolipoic acid coupled with poly(ethylene)glycol, multiple-thiols ligand greatly enhance the stability over a wide range of biological conditions while maintaining a small hydrodynamic radius.
• Ligands based on lipoic acid and related compounds which contain the 1 ,2-dithiolane anchoring unit, have become increasingly popular, owing to their ability to form robust capping monolayers on the surface of metal and semiconductor nanoparticles.
• the high stability of the layer arises from the presence of two efficient surface binding sites in each anchoring moiety, generated by the rupture of the S-S bond of the dithiolane moiety.
• the cleavage of the disulfide bond occurs spontaneously in the presence of a noble metal surface, while it has to be activated in the case of semiconductor surfaces.
• the dithiolane group is chemically reduced to bis-thiol.
• DHLA dihydrolipoic acid
• the lipoic acid reduction is commonly carried out using a solution of NaBH 4 (A.F. Wagner, E. Walton, G. E. Boxer, M. P. Pruss, F. W. Holly, K. Folkers, "Properties and Derivatives of a-Lipoic Acid", J. Am. Chem. Soc, 1956, 78, 5079-5081 ; C. Gunsalus, L. S. Barton, W. Gruber, "Biosynthesis and Structure of Lipoic Acid Derivatives", J. Am. Chem. Soc, 1956, 78, 1763-1766).
• the protocols currently available for the reduction of lipoic acid involve the breaking of S-S bond through reaction with NaBH 4 at low temperatures for several hours.
• the obtained product is then acidified and purified through extraction with water/toluene or water/chloroform (H. Mattoussi, J. M. Mauro, E.R. Goldman, G.P. Anderson, V.C. Sundar, F.V. Mikulec, M.G. Bawendi, "Self-Assembly of CdSe-ZnS Quantum Dot Bioconjugates Using an Engineered Recombinant Protein", J. Am. Chem. Soc, 2000, 122, 12142- 12150; H. T. Uyeda, I. L. Medintz, J. K. Jaiswal, S. M. Simon, H.
• Mattoussi "Synthesis of Compact Multidentate Ligands to Prepare Stable Hydrophilic Quantum Dot Fluorophores", J. Am. Chem. Soc, 2005, 127, 3870-3878; A. R. Clapp, E. R. Goldman, H. Mattoussi, “Capping of CdSe-ZnS quantum dots with DHLA and subsequent conjugation with proteins", Nat. Protoc, 2006, 1 , 1258-1266; B. C Mei, K. Susumu, I. L Medintz, H. Mattoussi, "Polyethylene glycol-based bidentate ligands to enhance quantum dot and gold nanoparticle stability in biological media", Nat. Protoc, 2009, 4,412-423).
• the reduced bis-thiol ligand needs to be stored in a refrigerator under inert atmosphere to prevent re-oxidation.
• NaBH 4 as a reducing agent is not possible; for example, with functional ligands with metal ion receptors, which are therefore sensitive to metal ions.
• Another type of ligand exchange involves the use of thiolated silanes. These ligands are directly absorbed on the QD surface after displacement of the native ligands (M. Bruchez Jr., M. Moronne, P. Gin, S. Weiss, A. P. Alivisatos, Science, 1998, 281, 2013-2016; D. Gerion, F. Pinaud, S. C. Williams, W. J. Parak, D. Zanchet, S. Weiss, A. P. Alivisatos, J. Phys. Chem. B, 2001 , 105, 8861 -8871 ).
• the addition of a base triggers the hydrolysis of the silanol groups with the formation of a silica-siloxane shell. Further addition of the siloxane precursor produces a much thicker shell with the aim of a better water compatibility.
• Ligands for quantum dots bearing a dithiolane group and functionalized with PEG are known. See for example the following literature.
• Ligands with a dithiolane group which can be used for quantum dots are also disclosed in FR2925492 and in WO2013025347.
• US6426513 discloses the superficial functionalization of semiconductor nanocrystals using ligand containing mono-thiols. As explained above, the use of mono-thiol ligands has some disadvantages in terms of stability of the nanocrystal, in particular in aqueous solution.
• US6369098 discloses methods for synthetizing dithiolane derivatives, which are ligands for certain cell receptors and are useful in the treatment of a series of disorders.
• US6649138 discloses a method for the water solubilization of semiconductor nanocrystals using amphiphilic polymers having a hydrophobic moiety able to interact with the nanocrystal through non-covalent bindings and a hydrophilic moiety able to promote the solubilization of the nanocrystals in a water environment.
• Said method of functionalization differs therefore from the method of ligand exchange, in particular since the interaction ligand-nanocrystal is of non-covalent type.
• the disclosed ligands are all polymers.
• US6319426 discloses a water-soluble semiconductor nanocrystal including a semiconductor nanocrystal core, a shell-layer overcoating the core comprising a semiconductor material, and an outer layer comprising a molecule having at least one linking group for attachment of the molecule to the overcoating shell-layer and at least one hydrophilic group, optionally spaced apart from the linking group by a hydrophobic region sufficient to prevent electron charge transfer across the hydrophobic region.
• ligands for the solubilization in water of the nanocrystals are reported, in particular ligands having thiols groups for the anchoring to the nanocrystals surface.
• lipoic acid is disclosed and it is reduced using NaBH 4 according to a method known in the art (C. Gunsalus, L. S. Barton, W. Gruber, "Biosynthesis and Structure of Lipoic Acid Derivatives", J. Am. Chem. Soc, 1956, 78, 1763-1766).
• the inventors of the present invention have found a method for the production of functionalized QDs, which involves the quick reduction of ligands based on dithiolane group, in particular lipoic acid, and their subsequent use in the functionalization of QDs through exchange with the native surface ligands.
• Said method is based on the use of a borohydride ion-exchange resin (N.M. Yoon, H.J. Lee, J.H. Ahn, J. Choi, "Selective reduction of alkyl halides with borohydride exchange resin-nickel acetate in methanol", J. Org. Chem., 1994, 59, 4687-4688) for the reduction of the dithiolane unit.
• US6888019 discloses a method of preparing a rhenium complex by use of a borohydride exchange resin, where the resin is used as a reducing agent for breaking the S-S bond of disulphide, thus converted into sulphide, which is then combined with rhenium.
• the obtained rhenium complexes are useful as radioactive pharmaceuticals.
• the compounds obtained by the disclosed method and their use are completely different from the compounds obtained in the present invention and their field of application.
• the strategy reported here is particularly appropriate for the activation of ligands that are sensitive to metal ions (e.g., because they contain metal ion receptors).
• the inventors of the present invention have found that the use of the method of the invention for the reduction of ligands allows for the production of QDs covered with a reduced ligand in the form of a carboxylate ion and a layer of countercations non- covalently bound to the carboxylate ion.
• NCs with a coating of cationic ions are also disclosed in the work of Kovalenko et al. ("Nanocrystal superlattices with thermally degradable hybrid inorganic-organic capping ligands", J. Am. Chem. Soc, 2010, 132, 15124-15126).
• NCs functionalized with metal chalcogenide complexes (MCCs) are turned from highly hydrophilic to nonpolar and lipophilic using cationic surfactant molecules which bind to the negatively charged surfaces forming dense hydrophobic monolayers.
• MCCs metal chalcogenide complexes
• Na + or NH 4 + counterions are partially replaced with different tertiary alkylammonium ions; other long-chain cationic surfactants can be used.
• Said cationic surfactants are therefore used to make the MCC-capped NCs compatible with common nonpolar molecules, therefore soluble in nonpolar solvents.
• the problem of solubility is therefore mentioned in this paper only with regard to non-polar solvents and the cationic molecules are used for making the NCs surface hydrophobic.
• the disclosed ligands are all inorganic.
• oligo(ethyleneglycol)-based molecular or polymeric ligands afford NCs that are soluble in various solvents depending on chain length (H. T. Uyeda, I. L. Medintz, J. K. Jaiswal, S. M. Simon, H. Mattoussi, "Synthesis of Compact Multidentate Ligands to Prepare Stable Hydrophilic Quantum Dot Fluorophores", J. Am. Chem. Soc, 2005, 127, 3870-3878; I. Yildiz, B. McCaughan, S. F. Cruickshank, J. F. Callan, F. M.
• the functionalization of QDs with different countercations allows for the modulation and control of the solubility of the QDs in aqueous solution and in other polar solvents.
• the functionalized QD has different solubility properties in different polar solvents depending on the type of countercations with which it is functionalized.
• the alkyl chains can be the same or different, linear or branched.
• the tetralkylammonium is selected from the group consisting of: tetramethylammonium (TMA + ), tetrabutylammonium (TBA + ), tetraethylammonium (TEA + ) and tetraoctylammonium (TOA + ).
• Said ligand comprises a moiety with a dithiolane group, binding the quantum dot, and a moiety with a salified acid group.
• Said salified acid group can be a carboxylate, a sulfonate or a phosphate group.
• it is a carboxylate group.
• said ligands are ligands based on lipoic acid.
• a method for manufacturing said quantum dot is also an object of the present invention. Said method comprises the following steps:
• step d adding quantum dots covered with hydrophobic ligands, said quantum dots being in a solid form or dissolved in a second solvent not miscible with said first solvent, to the solution of step d), thus obtaining a biphasic mixture;
• the quantum dots added in step e) are dissolved in a second solvent not miscible with said first solvent; a biphasic mixture is thus obtained.
• the quantum dots are transferred from said second solvent to the solution of step d), typically forming a suspension.
• the second solvent is then removed and the suspension is washed with said second solvent.
• the first solvent can be removed and the QDs isolated (step g).
• said quantum dots added in step e) are in a solid form.
• shaking of the mixture allows the transfer of the solid quantum dots to the solution containing the reduced, hydrophilic ligands, typically forming a suspension. Said suspension can then be washed with a second solvent not miscible with the first solvent of said solution. After separation of the solvents, the first solvent can be removed and QDs isolated (step g).
• Said method allows for the preparation of quantum dots covered with the desired hydrophilic ligands and countercations, which can be then solubilized in water or other polar solvent.
• polar solvents in which said QDs can be solubilized are water, methanol, acetone, acetonitrile.
• quantum dots with different characteristics of compatibility with polar solvents can be obtained.
• the QDs obtained with the method of the present invention maintain largely their optical properties and are stable in solution for a long time.
• quantum dots of the invention as luminescent probes/labels in biology and medical diagnostics, as components of photosensitizers for photodynamic therapy, in light absorbing materials for solar cells and in light emitting materials for lighting and display technologies is also within the scope of the present invention.
• the present invention will be now disclosed in detail also by means of examples. DESCRIPTION OF THE INVENTION
• quantum dot means a semiconductor nanocrystal with size-dependent optical and electronic properties.
• nanonocrystal is used in the present invention as a synonym of "quantum dot”.
• ligand means a molecule able to bind to the surface of a quantum dot.
• Figure 6 Absorption spectra of a lipoic acid/DHLA methanol solution before (full line) and after (dashed line) addition of TMAOH. Part (b) shows a magnification of the region of the S-S absorption band peaking at 330 nm.
• Figure 7 a) Absorption spectrum of a lipoic acid/DHLA solution soon after the addition of the base TMAOH (dashed line), and changes observed on stirring for up to 20 min (full line), b) Absorption spectra of lipoic acid before reduction (full line), and after reduction and base extraction (dashed line).
• Figure 8 Absorption spectrum of 1 .6x10 -2 M lipoic acid in methanol before (a) and after the addition of BH 4 ⁇ resin (2 equivalents) and 30 min stirring (b).
• Curve (c) is the spectrum obtained upon treating the mixture in (b) with NaOH (2 equivalents with respect to lipoic acid) and 30 min stirring.
• a quantum dot includes a "core" of one or more first semiconductor materials, which can be surrounded by a “shell” of a second semiconductor material.
• a semiconductor nanocrystal core surrounded by a semiconductor shell is referred to as a "core/shell” semiconductor nanocrystal.
• the core and/or the shell can be a semiconductor material including, but not limited to, those of the group ll-VI (e.g., ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe and the like) and lll-V (e.g., GaN, GaP, GaAs, GaSb, InN, InP, In As, InSb, AIAs, AIP, AlSb, AIS, and the like) and IV ( e.g., Ge, Si, Pb and the like) materials, and an alloy thereof, or a mixture, including ternary and quaternary mixtures, thereof.
• ll-VI e.g., ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe and the like
• lll-V
• the core can be synthesized using the published procedure developed by Peng and coworkers (Z. A. Peng, X. Peng, "Formation of High-Quality CdTe, CdSe, and CdS Nanocrystals Using CdO as Precursor", J. Am. Chem. Soc, 2001 , 123, 183-184).
• the shell overcoating reaction can be carried out using either the successive ion layer adsorption and reaction (SILAR) (J. J. Li, Y. A. Wang, W. Guo, J. C. Keay., T. D. Mishima, M.B. Johnson, X.
• SILAR successive ion layer adsorption and reaction
• Peng "Large-Scale Synthesis of Nearly Monodisperse CdSe/CdS Core/Shell Nanocrystals Using Air-Stable Reagents via Successive Ion Layer Adsorption and Reaction", J. Am. Chem. Soc, 2003, 125, 12567-12575) or the one-time-precursors-injection approach (M. A. Hines, P. Guyot-Sionnest, "Synthesis and Characterization of Strongly Luminescing ZnS-Capped CdSe Nanocrystals", J. Phys. Chem, 1996, 100, 468-471 ; B. O. Dabbousi, J. Rodriguez-Viejo, F. V.
• Core/shell semiconductor nanocrystals can be prepared in high-boiling point non- coordinating organic solvents using a two step approach in which a relatively thick shell is grown onto core nanocrystals synthesized in the first step.
• the resulting nanocrystals are covered with hydrophobic ligands, for example TOPO (tris- octylphosphineoxide), TOP (trioctylphosphine), OIA (oleic acid), ODA (octadecylamine) and/or HDA (hexadecylamine); they act as passivating hydrophobic surface agents, preventing particle aggregation.
• hydrophobic ligands for example TOPO (tris- octylphosphineoxide), TOP (trioctylphosphine), OIA (oleic acid), ODA (octadecylamine) and/or HDA (hexadecylamine); they act as passivating hydrophobic surface agents, preventing particle aggregation.
• CdSe, CdTe and CdS are quantum dot cores more suitable for the present invention.
• ZnS, CdS and ZnSe are quantum dot shells particularly suitable for the present invention.
• the QD core is CdSe and the shell is ZnS.
• the QD can vary in size and shell thickness depending on the number of shell monolayers.
• the number of shell monolayers is 3 or 5.
• the QDs are prepared in high-boiling point non- coordinating organic solvents using a two step approach in which a relatively thick ZnS shell is grown onto CdSe core nanocrystals synthesized in the first step.
• the resulting nanocrystals are covered with TOPO (tris-octylphosphineoxide) and TOP (trioctylphosphine), OIA (oleic acid), ODA (octadecylamine) and/or HDA (hexadecylamine).
• Said QDs are then used in step e) of the method of the invention to obtain the quantum dots of the invention.
• the quantum dot of the present invention is covered with hydrophilic ligands bearing a dithiolane group and a salified acid group and countercations.
• the ligands are ligands based on lipoic acid.
• the ligand is lipoic acid, which is reduced to di-hydro lipoic acid (DHLA) in order to bind to the quantum dot.
• DHLA di-hydro lipoic acid
• DHLA Lipoic acid Di-hydro lipoic acid
• the QD of the present invention is characterized in that it is soluble in polar solvent and, in particular, in that its solubility can be modulated by the type of surface functionalization. In fact, according to the countercation with which the QD surface is functionalized, the QD is endowed with specific characteristics of compatibility with different polar solvents.
• step c it is possible to tune the solubility of the QDs by changing the type of salt or base used in the extraction step (step c) of the method of the invention, in particular by changing the countercation of said salt or base.
• the method of the invention has in fact the further advantage to allow the modulation of the solubility of the QDs in different polar solvents by simply changing the kind of base (and thus of countercation) to be used in the method. This avoids the effort of synthesizing different QDs with different ligands according to the solvent, as done in the prior art.
• the use of the method of the invention does not substantially change or alter the properties of the functionalized quantum dots.
• the quantum dots of the present invention are obtained by the method above described, comprising the steps a)-g), which is also an object of the present invention. Said method will be now described more in detail.
• step a) of said method a resin loaded with BH 4 ⁇ is added to a solution of the ligand bearing a dithiolane group and a salifiable acid group in a first solvent.
• said first solvent is selected from the group consisting of methanol, ethanol or water.
• the molar ratio between the ligand and BH 4 ⁇ in step a) is preferably of 1 :2.
• the obtained mixture is stirred for a time sufficient for the ligand to go inside the resin, react and stick to the resin. Said stirring time is at least 30 min, preferably higher than 30 min.
• the resin loaded with BH 4 ⁇ is commercially available and can be purchased or otherwise prepared with methods known in the art. For example, it can be prepared starting from a commercially available anion-exchange resin loaded with anions CP, where said anions are subsequently exchanged with anions BH 4 ⁇ using an aqueous solution of NaBH 4 (N.M. Yoon, H.J. Lee, J.H. Ahn, J. Choi, "Selective reduction of alkyl halides with borohydride exchange resin-nickel acetate in methanol", J. Org. Chem., 1994, 59, 4678-4688). This second option is economically more advantageous.
• Said resin is preferably in the form of beads.
• step b) the solvent is removed and the resin with the attached ligand is obtained.
• Said resin can be optionally washed with the solvent, to remove the unreacted ligand and the hydrolyzed borohydride products.
• step c) a base or a salt thereof is added to the resin, preferably in a ratio ranging from 1 .2 to 4 equimolar with respect to the BH 4 ⁇ content of the resin.
• the mixture is stirred for a suitable time to extract the reduced ligand from the resin; preferably said time is 30 minutes.
• a base When a base is used, it is a Bronsted base. Preferably, it is a hydroxide.
• a salt is preferably a triflate (CF3SO3 “ ), a bromide (Br “ ) or a perchlorate (CIO 4 " ) salt.
• the cation of said base or salt thereof is selected from the group consisting of Na + , Li + , K + , Zn ++ , Fe ++ , Cu ++ , d-C 8 tetralkylammonium.
• the cation is a C-i-Cs tetraalkylammonium cation, it is preferably selected from the group consisting of tetramethylammonium (TMA + ), tetraethylammonium (TEA + ), tetra(n-butyl)ammonium (TBA + ) and tetra(n-octyl)ammonium (TOA + ).
• TBA + tetramethylammonium
• TEA + tetraethylammonium
• TSA + tetra(n-butyl)ammonium
• TOA + tetra(n-octyl)ammonium
• a more preferred cation is TBA + .
• step d The resin is then washed with the solvent (step d).
• said washing with the solvent can be repeated in order to recover as much reduced ligand as possible; all the solvent fractions are then joined together and the residual solid are discarded.
• step e the quantum dots covered with hydrophobic ligands, obtained as above described, are added to the solution of step d) containing the reduced ligand.
• the amount of the added QD in step e) may vary between 1/20000 and 1/30000 QD/ligand ratio, depending on the QD size.
• the quantum dots covered with hydrophobic ligands can be added to the solution of step d) in a solid form or dissolved in a second solvent not miscible with said first solvent.
• Said mixture is shaken to allow the transfer of the QDs to the solution containing the reduced ligand to allow exchange of the native hydrophobic ligands with the hydrophilic, reduced ligands (step f). It can be further stirred for a suitable time, preferably overnight, to allow a complete ligands exchange.
• the obtained QDs are then isolated (step g).
• Isolation of QDs can be done according to the general knowledge of the skilled person.
• the QDs are added in solid form, once they are transferred to the solution containing the reduced, hydrophilic ligands, typically a suspension is formed.
• the suspension can be washed with a second solvent not miscible with the first solvent of said solution in order to remove unreacted QDs and native hydrophobic ligands.
• the first solvent can be removed, preferably under reduced pressure, and QDs isolated.
• the QDs in case QDs are added dissolved in a second solvent, not miscible with the first solvent, the QDs, once transferred to the solution containing reduced, hydrophilic ligands, typically form a suspension.
• the non- miscible solvents are separated and the suspension can be treated as described above.
• said second solvent is hexane.
• Quantum dots covered with hydrophilic ligands and countercations according to the present invention are thus obtained.
• Said QDs can then be dissolved in water or in other polar solvent thus obtaining a solution of QDs.
• Said solution of QDs can be filtered to remove possible large aggregates.
• a syringe filter may be employed.
• Said solution can be further purified by removing the reduced ligand in excess with cycles of dilution/concentration.
• the number of said cycles is 3 and a centrifugal filter is preferably employed.
• the method of the invention above described is preferably carried out at a temperature ranging from 20 to 60°C.
• the concentration of the ligand in step a) is preferably lower than 50 mM.
• the QD/ligand ratio is preferably comprised between 1/20000 and 1/30000.
• the QD is functionalized with dihydrolipoic acid (DHLA), as the reduced ligand, and Na + as the countercation.
• DHLA dihydrolipoic acid
• the QD is a DHLA7Na + -coated core-shell CdSe- ZnS QD.
• the present invention also provides a method for controlling solubility of quantum dots characterized in that when the solvent is water the acid group of the ligand according to the invention is salified with a countercation selected from the group consisting of Na + , Li + , K + , TMA + , TEA + and TBA + .
• the present invention also provides a method for controlling solubility of quantum dots characterized in that when the solvent is dimethyl sulfoxide (DMSO) the acid group of the ligand according to the invention is salified with TBA + .
• DMSO dimethyl sulfoxide
• the present invention also provides a method for controlling solubility of quantum dots characterized in that when the solvent is methanol the acid group of the ligand according to the invention is salified with a countercation selected from the group consisting of K + , TMA + , TEA 4" , TBA + and TOA + .
• the present invention also provides a method for controlling solubility of quantum dots characterized in that when the solvent is acetonitrile the acid group of the ligand according to the invention is salified with a countercation selected from the group consisting of TEA 4" and TBA + .
• the present invention also provides a method for controlling solubility of quantum dots characterized in that when the solvent is acetone the acid group of the ligand according to the invention is salified with TBA + .
• the quantum dot of the present invention can be used as luminescent probe/label for in vitro biological applications; for example, for biochemical analysis.
• the quantum dots of the invention can be used as luminescent probes/labels in medical diagnostics and/or for medical imaging.
• the quantum dot can be used as a component of photosensitizers for photodynamic therapy.
• Photodynamic therapy is a form of phototherapy used for treating a variety of medical conditions, wherein light- sensitive compounds are exposed selectively to light.
• a photosensitizer comprising the quantum dot of the invention is also within the scope of the present invention.
• the quantum dots of the invention can also be included in light absorbing materials; said materials can be used, for example, for the manufacturing of solar cells.
• a light absorbing material comprising the quantum dot of the invention is also an object of the present invention.
• the quantum dots are included in light emitting material, which can be used for lighting apparatus and displays.
• light emitting material which can be used for lighting apparatus and displays.
• quantum dots reference can be made to Y. Shirasaki, G. J. Supran, M. G. Bawendi, V. Bulovic, "Emergence of colloidal quantum-dot light-emitting technologies", Nat. Photonics, 2013, 7, 13-23, and references therein.
• a light emitting material comprising the quantum dot of the invention is also an object of the present invention.
• the following examples will further illustrate the invention.
• the reduction of lipoic acid is achieved by stirring 5.5 mg of lipoic acid (2.66x1 CP 5 mol) with 19 mg of BH 4 ⁇ resin (2.7 mmol BH 4 ⁇ per g) in 500 ⁇ _ of methanol at 400 rpm for at least 30 min. A longer stirring time afforded a better reduction. During this time, the lipoic acid goes inside the resin, reacts and sticks into it.
• the mixture was further stirred overnight to allow complete exchange of the native hydrophobic ligands with the new hydrophilic ones.
• the borohydride-loaded resin was prepared following the protocols reported in literature (N. M. Yoon, H. J. Lee, J. H. Ahn, J. Choi, "Selective reduction of alkyl halides with borohydride exchange resin-nickel acetate in methanol", J. Org. Chem., 1994, 59, 4687-4688).
• the amount of BH 4 ⁇ loaded estimated by acid titration, is in agreement with that reported for a commercially available borohydride-loaded resin (Sigma-Aldrich, Borohydride on Amberlite ® IRA-400, Catalog Number: 328642). In all cases, we used a resin loaded with 2.7 mmol of BH 4 ⁇ per gram of resin.
• DHLA Na + -coated core-shell CdSe-ZnS QDs of various size and different shell thickness (3 monolayers and 5 monolayers) yielded clear water solutions which resulted to be stable for at least 3 months. Only a very minor shift in the absorption and emission peak wavelengths was observed with respect to the starting hydrophobic QDs, indicating that no aggregation takes place and the spectroscopic properties of the final products are preserved.
• the luminescence efficiency of the final QDs in aqueous solution is 30%-50% of that of the starting nanoparticles in organic solvent, as widely reported in literature (A. R.
• FIG. 1 shows the absorption and emission spectra of CdSe-3ZnS and CdSe-5ZnS orange capped with DHLA7Na + in water, compared with the same nanocrystals TOP/TOPO capped in CHCI3.
• the absorption spectrum of the QDs capped with DHLA7Na + is unchanged with respect to the TOP/TOPO capped QDs, thus confirming that the functionalized QDs are intact and their absorption properties are not modified.
• the emission intensity of the DHLA7Na + QDs is 30-50% lower than that of the TOP/TOPO capped QDs, as expected and already known in literature for QDs covered with hydrophilic ligands.
• DHLA7Na + capped QDs prepared as above described were evaluated.
• a dilute solution (130 nM) of the nanocrystals of Figure 1 b (CdSe-5ZnS QDs DHLA7Na + capped) in deionized water was stored in a refrigerator at 5°C, and the absorption and luminescence spectra were monitored over 3 weeks ( Figure 2).
• No precipitation was observed, although the emission quantum yield decreased from 0.081 to 0.05 during the first two weeks, in line with literature reports for DHLA-capped QDs (D. Liu, P. T. Snee, "Water-Soluble Semiconductor Nanocrystals Cap Exchanged with Metalated Ligands", ACS Nano, 2011 , 5, 546- 550).
• TBA + offers solubility in a wide range of solvents but in this case the QDs are less soluble in water, owing to the fact that the nanocrystals' surface bear long hydrophobic alkyl chains.
• TOA + -covered QDs are soluble only in methanol.
• FIG. 4 shows photographs of QDs capped with DHLA- TBA + in different solvents.
• the reduction of the lipoic acid was studied by following the change of the S-S absorption band at 330 nm.
• 2.5 ml_ of a methanol solution of lipoic acid (1 .6x10 -2 M) was placed in a spectrophotometric cell together with a certain amount of borohydride resin (2 equivalents of BH 4 -). The solution was stirred.
• the absorption changes are depicted in Figure 5.
• TMAOH tetramethylammonium hydroxide

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