Water-soluble fluorescent semiconductor nanocrystals

IS 2 228 107 T3

DESCRIPTION

Water soluble fluorescent semiconductor nanocrystals.

Field of the invention

The present invention relates to water-soluble nanocrystalline materials that emit energy in a narrow range of wavelengths. In particular, the invention relates to water-soluble semiconductor nanocrystals that emit light in the visible and infrared energy range.

Background of the invention

Semiconductor nanocrystals (also known as Quantum Dot ™ particles whose radii are smaller than the effective Bohr radius of the exciton are a class of materials that straddle the molecular forms and mass forms of matter. Quantum confinement of both the electron and the hole, in all three dimensions, leads to an increase in the effective band gap of the material by decreasing the size of the crystallite. Consequently, both the absorption and the optical emission of semiconductor nanocrystals shift towards the blue (higher energies) as the size of the nanocrystals becomes smaller.

Bawendi et al. Have described a process for preparing monodisperse semiconductor nanocrystals by pyrolysis of injected organometallic reagents in a hot coordinating solvent (Murray et al. (1993) J. Am. Chem. Soc. 115: 8706). This allows for temporally discrete nucleation and results in a controlled growth of macroscopic amounts of nanocrystals. Size-selective precipitation of crystallites from the growth solution can provide crystallites with even narrower size distributions. The narrow size distribution of semiconductor nanocrystals allows the possibility of light emission with narrow widths of spectral lines.

In an effort to improve the photoluminescent performance of semiconductor nanocrystals, the surface of the nanocrystal has been passivated by reaction of the surface atoms of the nanocrystal with passivating organic ligands, to eliminate energy levels on the surface of the crystallite that fall within the energy band prohibited from the interior mass. These surface energy states act as traps for electrons and holes that degrade the luminescence properties of the material. Such passivation produces an atomically abrupt increase in chemical potential at the interface of the semiconductor and the passivating layer (see, Alivisatos (1996) J. Phys. Chem. 100: 13226). Murray et al. (1993), above, describe CdSe nanocrystals terminated with organic moieties such as tri-n-octylphosphine (TOP) and tri-n-octylphosphine oxide (TOPO) with quantum yields as high as 20% in organic solvents such as toluene (see also Christopher Murray's doctoral thesis “Synthesis and Characterization of II-VI Quantum Dots and Their Assembly into 3-D Quantum Dot Superlattices” (1995) Massachusetts Institute of Technology; and Kuno et al. (1997) J. Phys Chem. 106 (23): 9869).

Although semiconductor nanocrystals prepared as described by Bawendi et al. Show close monodispersity, and thus high color selectivity, the luminescence properties of the material are process dependent. The stability of the photoluminescent property of the nanocrystal is a function of the nature of the passivating species that coats the external surface of the nanocrystal.

Known organically coated nanocrystals are not robust and show degradation of photoluminescent performance in solution. This is probably due to dissociation of the passivating layer from the surface of the nanocrystal, or degradation of the passivating layer which results in degradation of the semiconductor surface.

Passivation of semiconductor nanocrystals using inorganic materials has also been disclosed. Passivated particles with an inorganic coating are much more robust than organically passivated particles, and exhibit greater tolerance to the processing conditions required for incorporation into devices. Previously disclosed inorganically passivated semiconductor nanocrystal particle structures include CdS-terminated CdSe and CdSe-terminated CdS (Than et al. (1996) J. Phys. Chem. 100: 8927); ZnS grown on CdS (Youn et al. (1988) J. Phys. Chem. 92: 6320); ZnS on CdSe and the reverse structure (Kortan et al. (1990) J. Am. Chem. Soc. 112: 1327); Zns-terminated CdSe nanocrystals (Hines et al. (1996) J. Phys. Chem. 100: 468); ZnSe-terminated CdSe nanocrystals (Danek et al. (1996) Chem. Materials 8: 173); and SiO₂ on Si (Wilson et al. (1993) Science 262: 1242).

Kortan et al. (1990), supra, describes a ZnS-terminated CdSe nanoparticle having a layer of thiophenyl groups attached to the outer surface. The thiophenyl groups were used to passivate the surface and to allow clusters to be isolated in powder form. Lawless et al. (1995) J. Phys. Chem. 99: 10329 reported the preparation of semiconductor CdS nanocrystals terminated with bifunctional mercaptocarboxylic acids HS (CH₂)_nCOOH, where n is 1 equal to 3. TiO particles were attached₂ to CdS nanocrystals through the carboxylic acid functional group of the terminator bifunctional moiety, in order to promote interparticle electron transfer between uneven semiconductor particles.

The semiconductor nanocrystals described above are soluble or dispersible in organic solvents.

ES 2 228 107 T3 only, such as hexane or pyridine. Many applications that rely on fluorescent emission from semiconductor nanocrystals require that the semiconductor nanocrystals be soluble in water.

Many disclosed water soluble semiconductor nanocrystals suffer from significant disadvantages that limit their general applicability. For example, Spanhel et al. (1987) J. Am. Chem. Soc. 109: 5649, describes a CdS-terminated Cd (OH) sol₂; however, the photoluminescent properties of the sun were pH dependent. The sol could only be prepared in a very narrow range of pH (pH 8 to 10), and showed a narrow band of fluorescence only at a pH greater than 10. Such dependence on pH greatly limits the usefulness of the material. In particular, it is not suitable for use in biological systems.

Other groups have replaced the organic passivating layer of the semiconductor nanocrystal with water-soluble residues; however, the resulting derivatized semiconductor nanocrystals are not very luminescent. Short chain thiols, such as 2-mercaptoethanol and 1-thioglycerol, have been used as stabilizers in the preparation of water soluble CdTe nanocrystals. See, Rogach et al. (1996) Ber. Bunsenges. Phys. Chem. 100: 1772 and Rajh et al. (1993) J. Phys. Chem. 97.11999. Other more exotic terminator compounds have been disclosed, with similar results. See, Coffer et al. (1992) Nanotechnology 3:69, which describes the use of deoxyribonucleic acid (DNA) as a terminator compound. In all of these systems, the coated semiconductor nanocrystals were not stable, and the photoluminescent properties degraded over time.

The unavailability of aqueous solutions or suspensions of semiconductor nanocrystals with intense photoluminescent emissions limits their application in a variety of water-based applications, such as biological applications. Furthermore, aqueous solutions can often be very aggressive chemical systems, and many of the known water soluble semiconductor nanocrystal systems degrade, primarily by photoanodic decomposition at the semiconductor surface interface, during prolonged exposures in water.

Thus, there is still a need for water soluble semiconductor nanocrystals that can be prepared as robust, stable suspensions or solutions in aqueous media. There is also a need for water soluble semiconductor nanocrystals intended to emit energy with high quantum efficiencies, possessing a narrow particle size distribution (and thus having a narrow spectral range of photoluminescence).

Summary of the invention

A primary objective of the invention is to solve the aforementioned needs in the art.

Another objective of the invention is to provide water soluble semiconductor nanocrystals that overcome the limitations of the prior art and that show high quantum yields with photoluminescence emissions of high spectral purity.

A further objective of the present invention is to provide a semiconductor nanocrystal that is easily soluble in aqueous systems and that demonstrates chemical and electronic stability in those systems.

A further object of the invention is to provide a water soluble semiconductor nanocrystal, derivatized to provide a binding or coupling ability.

In one aspect of the invention, a water soluble semiconductor nanocrystal for emitting energy is provided. According to the present invention, a water soluble semiconductor nanocrystal capable of emitting energy is provided, comprising:

a semiconductor nanocrystal core exhibiting a selected energy gap;

a crust layer that covers the nucleus of the semiconductor nanocrystal, the crust comprising a semiconductor material that presents a band gap of energy greater than that of the nucleus;

an outer layer comprising a molecule having a first part comprising at least one linking group for attachment to the nanocrystal, and a second part comprising at least one hydrophilic group. The nanocrystal includes a semiconductor nanocrystal core exhibiting a selected energy gap, coated with a crust layer of a material exhibiting an energy gap greater than that of the core and with appropriate band differences. The water soluble nanocrystal further comprises an outer layer on the outer surface of the coating layer. The outer layer includes a molecule that has at least one linking group for attaching the molecule to the coating layer, and at least one hydrophilic group optionally separated from the linking group by a sufficient hydrophobic region to minimize the transfer of electronic charge through the hydrophobic region.

The outer layer of the nanocrystal can comprise an organic molecule. The organic molecule may comprise moieties selected to provide solubility in an aqueous medium, such as a long chain hydrocarbon ending in a moiety that exhibits affinity for an aqueous medium, and a moiety that demonstrates affinity for the surface.

ES 2 228 107 T3 of semiconductor nanocrystal. The affinity for the nanocrystal surface promotes the coordination of the organic molecule to the outer surface of the semiconductor nanocrystal, and the rest with affinity for the aqueous medium stabilizes the suspension of the semiconductor nanocrystal.

In a preferred embodiment, the molecule has the structural formula (I)

H_zX¹((CH₂)_nCO₂H)_Y (I) and you leave it, in which X¹ is N, P or O = P; n is greater than or equal to 6; yzey are selected to satisfy the valence requirements of X¹.

In other preferred embodiments, the molecule has the structural formula (II)

<img file="ES2228107T3_D0001.tif" />

wherein: X and X 'are the same or different, and are selected from the group consisting of S, N, P, or O = P; Y is a hydrophilic moiety; and Z is absent or is a hydrophobic region having a backbone of at least six atoms. X and X 'may include other substituents to satisfy valence requirements, such as, for example, amines, thiols, phosphines, and phosphine oxides, substituted with hydrogen or other organic moieties. Furthermore, the bridging atoms X and X 'can be selected to form a 5- to 8-membered ring with coordination to the semiconductor surface. The bridging atoms are typically carbon, but can be other elements such as oxygen, nitrogen, and sulfur. The group Y can be any charged or polar group, such as a carboxylate, a sulfonate, a phosphate, a polyethylene glycol or other polyol, and an ammonium salt, for example, carboxylate (-CO₂ ), sulfonate (SO₃ ), hydroxide (OH), alkoxides, ammonium salts (-NH₄⁺), and phosphate (-PO4 ²) and phosphonate (-PO3 ²), and the like. Typically Z is an alkyl group or an alkenyl group, but can also include other atoms, such as carbon and nitrogen. The Z group can be further modified as described herein to provide attractive interactions with neighboring ligands.

In yet another preferred embodiment, the molecule has the structural formula (III):

<img file="ES2228107T3_D0002.tif" />

wherein: X, X 'and X "are the same or different, and are selected from the group consisting of S, N, P or O = P; Y is a hydrophilic moiety; and Z is a hydrophobic region having a backbone of at least six atoms. Groups X, X 'and X "may include other substituents in order to satisfy valence requirements, such as, for example, amines, thiols, phosphines and phosphine oxides, substituted with hydrogen or other organic moieties. Furthermore, the bridging atoms X, X 'and X "can be selected to form a 5- to 8-membered ring with coordination to the semiconductor surface. The bridging atoms are typically carbon, but can be other elements such as oxygen, nitrogen, and sulfur. The group Y can be any charged or polar group, such as a carboxylate, a sulfonate, a phosphate, a polyethylene glycol or other polyol, and an ammonium salt, for example, carboxylate (-CO2), sulfonate (SO₃ ), hydroxide (-OH), alkoxides, ammonium salts (-NH₄⁺), and phosphate (-PO4 ²) and phosphonate (-PO3^-2), and the like. Typically Z is an alkyl group or an alkenyl group, but can also include other atoms, such as carbon and nitrogen. Additionally Z can be modified as described herein to provide attractive interactions with neighboring ligands.

In other preferred embodiments, the molecule has the structural formula (IV):

(R¹) a - R² - [(R³) b (R⁴)_c]_d (IV)

ES 2 228 107 T3 in which:

R¹ is selected from the group consisting of heteroalkyl, heteroalkenyl, heteroalkynyl, -OR, SR, -NHR, -NR'R ", -N (O) HR, -N (O) R'R", -PHR, -PR 'R ”, -P (NR'R”) NR'R ”, -P (O) R'R”, -P (O) (NR'R ”) NR'R”, -P (O) (OR ') OR ", -P (O) OR, -P (O) NR'R", -P (S) (OR') R ", and -P (S) OR, where R, R 'and R "are independently selected from the group consisting of H, a branched or unbranched alkyl, a branched or unbranched alkenyl, a branched or unbranched alkynyl, a branched or unbranched heteroalkyl, a branched or unbranched heteroalkenyl, and a branched or unbranched heteroalkynyl, provided that when a is greater than 1, the R groups¹ they can be the same or different or they can be linked to form a cycloalkyl, cycloalkenyl, heterocycle, aryl, six, seven, eight, nine or ten membered heteroaryl, or a six to thirteen membered crown ether or heterocorone ether;

R² is selected from a link (i.e. R² is absent), a branched or unbranched alkylene, a branched or unbranched alkenylene, a branched or unbranched heteroalkylene, a branched or unbranched heteroalkenylene, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocycle, aryl and heteroaryl;

R³ is selected from a branched or unbranched alkylene, a branched or unbranched alkenylene, a branched or unbranched heteroalkylene, a branched or unbranched heteroalkenylene, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocycle, aryl, and heteroaryl;

R⁴ is selected from the group consisting of hydrogen, a carboxylate, a thiocarboxylate, an amide, an imide, a hydrazine, a sulfonate, a sulfoxide, a sulfone, a sulfite, a phosphate, a phosphonate, a phosphonium, an alcohol, a thiol, an amine, an ammonium, an alkylammonium, a nitrate, a sugar moiety, and a five-, six-, seven-, eight-, nine-, or ten-membered cycloalkenyl, cycloalkynyl, heterocycle, aryl, or heteroaryl;

a is equal to 1, 2, 3 or 4;

b is equal to 0, 1, 2 or 3;

c is equal to 0, 1, 2 or 3; and d is equal to 0, 1, 2 or 3, where, when d is equal to 2 or 3, the R groups³ they can be the same or different or they can be linked together to form a five, six, seven, eight, nine or ten membered cycloalkyl, cycloalkenyl, heterocycle, aryl or heteroaryl.

Preferably, R¹ it is a thiol (eg, -SH), a phosphine, a phosphine oxide, or an amine (eg, -NH2, -NHR or -NRR ').

Preferably, R² it contains between 6 and 20 atoms. More preferably, R² is a linear alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene or heteroalkynylene containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 atoms, or a cycloalkyl or heterocycle containing 5 or 6 atoms.

Preferably, when b equals 1, 2, or 3, R³ it contains between 6 and 20 atoms. More preferably, R³ is a linear alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene or heteroalkynylene containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 atoms, or a cycloalkyl or heterocycle containing 5 or 6 atoms.

Preferably, R⁴ is a carboxylate (-COO), a phosphonate (-PO₃ ), a sulfonate (-SO₃ ) or an ammonium (-N⁺HRR ').

In yet another preferred embodiment of the invention, the molecule has the structural formula (V):

- | Y '(R) |. - R² | X '(R⁴) |. (V) in which the hanging groups R¹ and R⁴, and the rest R², are as defined above; X² and Y² are the same or different, and are monomeric units selected from the group consisting of acrylate, styrene, imide, acrylamide, ethylene, vinyl, diacetylene, phenylene-vinylene, amino acid, sugar, sulfone, pyrrole, imidazole, thiophene and ether, and m ' and n 'are selected in relation to the number of coordination sites available on the surface of the semiconductor nanocrystal. It is desirable that m 'is not greater than the number of available coordination sites, and preferably it is not greater than about a quarter of the available coordination sites. In particular, m 'is in the range from about 3 to about 100. The value of n' is typically chosen to be in proportion to the value of m '. Thus, it is desirable that n 'is not greater than the number of available coordination sites, and preferably it is not greater than about a quarter of the available coordination sites. In particular, n 'is in the range of 3 to 100. The molecule can be a block copolymer, in which a first block is provided that includes a pendant group capable of functioning as a linking moiety, Y. A second block is provided that includes a pendant group capable of functioning as a hydrophilic group, X. The polymer block serves as a hydrophilic region. In a preferred embodiment, the molecule has the formula

IS 2 228 107 T3

<img file="ES2228107T3_D0003.tif" />

wherein the Xs are the same or different, and are elements selected from the group of S, N, P, or O = P; and the Y's are the same or different, and are hydrophilic moieties, such as carboxylates, sulfonates, phosphates, phosphonates, polyethylene glycol, ammonium salt, and the like. Group X may include other substituents in order to satisfy valence requirements, such as, for example, amines, thiols, phosphine, and phosphine oxides, substituted with hydrogen or other organic moieties. The terminal groups R and R 'can be any residue, including hydrogen. In particular, it is desirable that R be a polar moiety due to its proximity to the hydrophilic block. Similarly, it is desirable for R 'to be a nonpolar moiety due to its proximity to the hydrophobic block. Indices m and n are selected in relation to the number of coordination sites available on the surface of the semiconductor nanocrystal. It is desirable that m is not greater than the number of available coordination sites, and preferably that it is not greater than one quarter of the available coordination sites. In typical applications, m is in the range of 3 to 100. The value of n is typically chosen to be in proportion to the value of m. Thus, it is desirable that n is not greater than the number of available coordination sites, and preferably not greater than one quarter of the available coordination sites. In typical applications, n is in the range of about 3 to 100.

Without being bound by theory, the inventors believe that the coordination of the molecule having the structural formula (IV) to the coated nanocrystal occurs between the surface residues on the nanocrystal and the R moiety.¹ of the molecule.

In another preferred embodiment, the water-solubilizing outer layer can comprise a homogeneous population of molecules having the structural formulas (I), (II), (III), (IV) or (V), a mixed population of molecules of any structural formula, that is, a mixed population of molecules which all have the structural formula (I), (II), (III), (IV) or (V), or a mixed population of molecules that have a combination of two or more of the structural formulas (I), (II), (III), (IV) and (V).

In another aspect of the invention, a water-soluble semiconductor nanocrystal is provided in which the water-solubilizing layer is a bilayer, a first bilayer layer exhibiting affinity for the coating layer, and a second bilayer layer exhibiting a hydrophobic region adjacent to the first layer and ending in a hydrophilic group. The bilayer may include a lyophilic coordination molecule used in the manufacture of the semiconductor nanocrystal, as the first layer, and a surfactant as the second layer.

These and other embodiments of the present invention will readily occur to one skilled in the art in light of the description herein.

Brief description of the drawings

The invention is described with reference to the figures, which are presented for illustrative purposes only, and in which:

Figure 1 is a schematic illustration of the water soluble nanocrystal of the invention;

Figure 2 is a schematic illustration of various alternative embodiments of the water soluble layer of the nanocrystal;

Figure 3 is an illustration of a water soluble nanocrystal of the invention having a crosslinked hydrophilic hydrocarbon backbone;

Figure 4 is an illustration of a water soluble nanocrystal of the invention comprising a polymethacrylate region;

Figure 5 is a schematic illustration of a bilayer type water soluble nanocrystal of the invention; and Figure 6 is an illustration of the displacement reaction used in the formation of the water soluble nanocrystal of the invention.

IS 2 228 107 T3

Detailed description of the invention

Definitions and nomenclature

Before the present invention is described and explained in detail, it should be understood that this invention is not limited to specific assay formats, materials, or reagents, as such, of course, can vary. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

It should be noted that, as used in the specification and appended claims, the singular forms "a" and "the" include plural referents, unless the context clearly dictates otherwise. Thus, for example, the reference to "a nanocrystal" includes more than one nanocrystal; reference to "an outer layer" includes more than one of said outer layer; and the like.

In this specification, and in the claims that follow, reference will be made to a number of terms that will be defined to mean the following:

"Quantum dot ™ particles" are a semiconductor nanocrystal with size-dependent electronic and optical properties. In particular, the energy gap of a semiconductor nanocrystal varies with the diameter of the crystal.

"Semiconductor nanocrystal" includes, for example, inorganic crystallites between about 1 nm and about 1000 nm in diameter, preferably between about 2 nm and about 50 nm, more preferably about 5 nm to about 20 nm (such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nm), which include a "core" of one or more first semiconductor materials, and that it can be surrounded by a "shell" of a second semiconductor material. A core of the semiconductor nanocrystal surrounded by a semiconductor shell is referred to as a "core / shell" type semiconductor nanocrystal. The surrounding "shell" material will preferably have a bandgap greater than the bandgap of the core material, and can be chosen to have an atomic spacing close to that of the “core” substrate. The core and / or shell can be a semiconductor material, including, but not limited to, those Group II-Vl materials (e.g., ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe and the like) and III-V (for example, GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP, AlSb, AlS, and the like) and IV (for example, Ge, Si, Pb and the like), and an alloy thereof, or a mixture, including ternary and quaternary mixtures thereof.

A semiconductor nanocrystal is optionally surrounded by a "coating" of an organic capping agent. The organic capping agent can be any number of materials, but has an affinity for the surface of the semiconductor nanocrystal. In general, the capping agent can be an isolated organic molecule, a polymer (or a monomer for a polymerization reaction), an inorganic complex, and an extended crystalline structure. The coating is used to provide solubility, for example, the ability to homogeneously disperse a coated semiconductor nanocrystal in a chosen solvent, to provide functionality, bonding properties, or the like. In addition, the coating can be used to tailor the optical properties of the semiconductor nanocrystal.

"Quantum yield", as used herein, means the ratio of emitted to absorbed photons, eg, the quantum yield of photoluminescence.

In other embodiments of the invention, the coated nanocrystal is characterized in that the nanocrystal exhibits a deviation of less than 10% rms (root mean square), and preferably less than 5% rms, of the core diameter. Thus, the term "monodisperse particles" includes a population of particles in which the population of particles deviates less than 10% rms in diameter, and preferably less than 5% rms. The nanocrystal in an aqueous medium preferably shows photoluminescence having quantum yields greater than 10%, and most preferably in the range of about 10% to 30%.

The term "alkyl" as used herein includes a reference to a branched or unbranched saturated hydrocarbon group of 1 to 100 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl , isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl, and the like. The term "lower alkyl" includes an alkyl group of 1 to 20 carbon atoms, preferably 6 to 20 carbon atoms.

The term "alkylene", as used herein, includes a reference to a difunctional, saturated, branched or unbranched hydrocarbon chain containing from 1 to 100 carbon atoms, and includes, for example, methylene (-CH₂-), ethylene (-CH₂-CH₂-), propylene (-CH₂-CH₂-CH₂-), 2-methylpropylene (-CH₂-CH (CH₃) -CH₂-), hexylene (- (CH₂)₆-), and the like. "Lower alkylene" includes an alkylene group of 1 to 20, more preferably 6 to 20, carbon atoms.

The term "alkenyl" as used herein includes a reference to a branched or unbranched hydrocarbon group, of 2 to 100 carbon atoms, containing at least one carbon-carbon double bond, such as ethenyl, n -propenyl, isopropenyl, n-butenyl, isobutenyl, t-butenyl, octenyl, decenyl, tetradecenyl, hexa

ES 2 228 107 T3 decenyl, eicosenyl, tetracosenyl and the like. The term "lower alkenyl" includes an alkenyl group of 2 to 20 carbon atoms, preferably 6 to 20 carbon atoms, which contains a -CsC- bond.

The term "alkenylene" includes a reference to a difunctional branched or unbranched hydrocarbon chain containing from 2 to 100 carbon atoms and at least one carbon-carbon double bond. "Lower alkenylene" includes an alkenylene group of 2 to 20, more preferably 6 to 20, carbon atoms, which contains a carbon-carbon double bond.

The term "alkynyl", as used herein, includes a reference to a branched or unbranched hydrocarbon group, of 2 to 100 carbon atoms, containing at least one -C = C- bond, such as ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl, t-butynyl, octynyl, decynyl, and the like. Preferred alkynyl groups herein contain from 6 to 20 carbon atoms. The term "lower alkynyl" includes an alkynyl group of 2 to 10 carbon atoms, and a -C ^ C- bond.

The term "alkynylene" includes a reference to a difunctional branched or unbranched hydrocarbon chain containing from 2 to 100 carbon atoms and at least one carbon-carbon triple bond. "Lower alkynylene" includes an alkynylene group of 2 to 10 carbon atoms, which contains a -C ^ C- bond.

Optionally, an alkyl, alkylene, alkenyl, alkenylene, alkynyl or alkynylene chain may contain 1 to 6 linkages selected from the group consisting of -O-, -S- and -NR- in which R it is hydrogen, lower alkyl or lower alkenyl.

The terms "heteroalkyl", "heteroalkylene", "heteroalkenyl", "heteroalkenylene", "heteroalkynyl" and "heteroalkynylene" include a reference to alkyl, alkylene, alkenyl, alkenylene, alkynyl and alkynylene groups, respectively, in which one or more Carbon atoms have been replaced, for example, with nitrogen, sulfur or oxygen atoms.

"Alkoxy" includes a reference to the group -OR, where R is an alkyl radical as defined above. Examples of an alkoxy radical include, but are not limited to, methoxy, ethoxy, isopropoxy, and the like.

"Alkylamino" includes a reference to a radical -NHR, where R is an alkyl radical as defined above. Examples of alkylamino radicals include, but are not limited to, methylamino, (1-ethylethyl) amino, and the like.

"Alkylthio" includes a reference to a radical -SR where R is an alkyl radical as defined above. Examples of alkylthio radicals include, but are not limited to, methylthio, butylthio, and the like.

"Dialkylamino" includes a reference to a radical -NR'R ", where R 'and R" are each independently alkyl radicals as defined above. Examples of dialkylamino radicals include, but are not limited to, dimethylamino, methylethylamino, diethylamino, di (1-methylethyl) amino, and the like.

"Hydroxyalkyl" includes a reference to an alkyl radical as defined above, substituted with one or more hydroxy groups. Examples of hydroxyalkyl radicals include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl, 1- (hydroxymethyl) -2 -hydroxyethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyl, and 2- (hydroxymethyl) -3-hydroxypropyl, and the like.

The term "acyl" as used herein includes a reference to an alkyl group linked through a - (CO) - bond. The term "lower acyl" includes an acyl group in which the alkyl group, attached through a carbonyl bond, is a lower alkyl group.

The term "sugar moiety" includes a reference to monosaccharides, disaccharides, polysaccharides, and the like. The term "sugar" includes those moieties that have been modified, for example, in which one or more hydroxyl groups are substituted with halogen, alkoxy moieties, aliphatic groups, or functionalized as ethers, amines, or the like. Examples of modified sugars include: those containing a lower alkoxy group in place of a hydroxyl moiety, ie, a- or / -glycosides such as methyl-aD-glucopyranoside, methyl-jd-D-glucopyranoside, and the like; those that have been reacted with amines, ie, N-glycosylamines or N-glycosides such as N- (aD-glucopyranosyl) methylamine; those containing acylated hydroxyl groups, typically 1 to 5 lower acyl groups; those containing one or more carboxylic acid groups, for example, D-gluconic acid or the like; and those containing free amine groups such as D-glucosamine, D-galactosamine, N-acetyl-D-glucosamine, or the like. Examples of preferred saccharides are glucose, galactose, fructose, ribose, mannose, arabinose, xylose. Examples of polysaccharides are dextran and cellulose.

"Aryl" includes reference to a monovalent aromatic hydrocarbon radical consisting of one or more fused rings in which at least one ring is aromatic in nature, which may be optionally substituted with one or more of the following substituents: hydroxy, cyano, alkyl, alkoxy , thioalkyl, halo, haloalkyl, hydroxyalkyl, nitro, amino, alkylamino, and dialkylamino, unless otherwise indicated.

ES 2 228 107 T3 "Heteroaryl" includes a reference to a monovalent aromatic carbocyclic radical having one or more rings incorporating one, two or three ring heteroatoms (chosen from nitrogen, oxygen or sulfur), which may be optionally substituted with one or more of the following substituents: hydroxy, cyano, alkyl, alkoxy, thioalkyl, halo, haloalkyl, hydroxyalkyl, nitro, amino, and alkylamino and dialkylamino, unless otherwise indicated.

"Cycloalkyl" includes a reference to a monovalent saturated carbocyclic radical consisting of one or more rings, which may be optionally substituted with one or more of the following substituents: hydroxy, cyano, alkyl, alkoxy, thioalkyl, halo, haloalkyl, hydroxyalkyl, nitro , amino, alkylamino, and dialkylamino, unless otherwise indicated.

"Cycloalkenyl" includes reference to a monovalent unsaturated carbocyclic radical consisting of one or more rings and containing one or more carbon-carbon double bonds, which may be optionally substituted with one or more of the following substituents: hydroxy, cyano, alkyl, alkoxy, thioalkyl, halo, haloalkyl, hydroxyalkyl, nitro, amino, alkylamino, and dialkylamino, unless otherwise indicated.

"Cycloalkynyl" includes reference to a monovalent unsaturated carbocyclic radical consisting of one or more rings and containing one or more carbon-carbon triple bonds, which may be optionally substituted with one or more of the following substituents: hydroxy, cyano, alkyl, alkoxy, thioalkyl, halo, haloalkyl, hydroxyalkyl, nitro, amino, alkylamino, and dialkylamino, unless otherwise indicated.

"Heterocyclic" includes reference to a monovalent saturated carbocyclic radical, consisting of one or more rings, incorporating one, two or three heteroatoms (chosen from nitrogen, oxygen or sulfur), which may be optionally substituted with one or more of the following substituents: hydroxy, cyano, alkyl, alkoxy, thioalkyl, halo, haloalkyl, hydroxyalkyl, nitro, amino, alkylamino, and dialkylamino, unless otherwise indicated.

The term "crown ether" includes reference to a mono-, di-, trivalent, or higher multivalent radical (eg, 4, 5,6, 7, or 8), of a saturated unbranched heterocyclic molecule. Crown ethers are typically referred to as "x crown y" or "xCy", where x represents the total number of atoms in the molecule, and y represents the number of heteroatoms in the molecule. Thus, for example, "12 crown 4" is a crown ether containing 12 atoms, 4 of which are heteroatoms, and 18C6 is a crown ether containing 18 atoms, 6 of which are heteroatoms. Preferred heteroatoms are O, S, and N, and, in any particular crown ether, the heteroatoms can be the same or different. A "heterocrown ether" is a crown ether in which the heteroatoms are different. Preferred crown ethers are six to thirty membered crown or heterocorone ethers, 8C4, 9C3, 12C4, 15C5, 18C6 and 20C8 are most preferred, and 12C4 and 18C6 are even more preferred.

"Optional" or "optionally" means that the event or circumstance described below may or may not occur, and that the description includes cases in which such event or circumstance occurs, and cases in which it does not. For example, the phrase "optionally substituted alkylene" means that an alkylene moiety may or may not be substituted, and that the description includes both unsubstituted alkylene and substituted alkylene, and the like.

The present invention relates to water soluble semiconductor nanocrystals that are highly luminescent and stable in aqueous solutions. The nanocrystal is schematically represented in Figure 1. A semiconductor nanocrystal 10 is coated with an outer layer 14 that makes the crystal soluble in water. The outer layer 14 is further selected to maintain the luminescent properties of the nanocrystal, and to improve the strength of the nanocrystal in aqueous solutions. An optional coating layer 12 can be used to coat the semiconductor nanocrystal prior to application of the outer layer 14. The outer layer includes a molecule 15 having at least one linking group 16 for binding of the molecule to the coating layer , and at least one hydrophilic group 20 separated from the linking group by a hydrophobic region 18 sufficient to prevent transfer of electronic charge along the hydrophobic region. Note that the hydrophilic group 20 is depicted, for convenience, as a negative charge in Figure 1; however, the group can be positively charged, or polar neutral.

The nanocrystal includes a semiconductor nanocrystal that exhibits quantum confinement effects in its luminescent properties. These nanocrystals are known as "Quantum Dot ™ particles". When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the band gap of the semiconductor material used in the semiconductor nanocrystal. In quantum confined particles, the band gap is a function of the size of the nanocrystal.

Upon exposure to a light source, the semiconductor nanocrystal emits energy of a wavelength characteristic of its composition and size. The water soluble layer of the invention can be used with nanocrystals having various combinations of core and nanocrystal coating. The invention allows the preparation of a variety of water-soluble nanocrystals that have a very narrow distribution of particle sizes, and that show improvements in color purity and intensity of their photoluminescent emissions, as well as demonstrate strength and stability in suspensions or water-based solutions. Most group IIVI, III-V and IV semiconductors have been prepared as quantum-sized particles, and show quantum confining effects on their physical properties, and can be used in water-soluble nanocrystals of the invention. Exemplary materials suitable for use as semiconductor nanocrystal cores include CdS, CdSe, CdTe, ZnS, ZnSe, ZnTe, MgTe, GaAs, GaP, GaSb, GaN, HgS, HgSe, HgTe, InAs, InP, InSb, InN, AlAs, AlP, AlSb, AlS, PbS, PbSe, Ge, Si, and

ES 2 228 107 T3 alloys thereof, or a mixture thereof, including ternary and quaternary mixtures thereof.

Semiconductor nanocrystals are characterized by their uniform nanometric size. By "nano" size is meant less than about 150 Angstroms (zA.), And preferably in the range of 15 to 150 A. The nanocrystal is also substantially monodisperse in the broad range of sizes given above. By monodisperse, as that term is used herein, it is meant a colloidal system in which the suspended particles are substantially identical in size and shape. For the purposes of the present invention, "monodisperse particles" means that at least 60% of the particles fall within a specific range of particle sizes. In preferred embodiments, the monodisperse particles deviate less than 10% rms in diameter, and preferably less than 5%. Monodisperse semiconductor nanocrystals have been described in detail in Murray et al. (1993), above, the thesis of Murray (1995), above, and Kuno et al., Above.

In preferred embodiments, the semiconductor nanocrystal has a cladding crust-like layer. On the surface of the semiconductor nanocrystal, surface defects can result in electron traps, or holes that degrade the electrical and optical properties of the semiconductor nanocrystal. An insulating layer on the surface of the semiconductor nanocrystal provides an atomically abrupt jump in chemical potential at the interface, eliminating energy states that can serve as traps for electrons and holes. This results in greater efficiency in the luminescent process.

Suitable materials for the clad shell layer include semiconductors that have a higher energy gap than semiconductor nanocrystal. In addition to having a higher energy gap than semiconductor nanocrystals, suitable materials for the clad shell layer must have a good separation between the conduction and valence bands relative to the semiconductor nanocrystal. Thus, the conduction band is desirably larger, and the valence band is desirably smaller than that of the semiconductor nanocrystal core. Thus, the core can be coated with a shell material comprising ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgS, MgSe, GaAs, GaN, GaP, GaAs, GaSb, HgO, HgS, HgSe, HgTe, InAs, InN, InP, InSb, AlAs, AlN, AlP, AlSb, an alloy thereof, or a mixture thereof, including ternary and quaternary mixtures thereof. Preferably, the energy gap of the clad shell is greater than that of the core. For semiconductor nanocrystals that emit energy in the visible (eg CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, GaAs) or near IR (eg InP, InAs, InSb, PbS, PbSe), you can use a material that exhibits an energy gap in the ultraviolet regions. Exemplary materials include ZnS, GaN, and magnesium chalcogenides, for example MgS, MgSe, and MgTe. For semiconductor nanocrystals that emit in the near IR, materials that have an energy gap in the visible, such as CdS or CdSe, can also be used. The skin shell layer can include up to eight monolayers of the semiconductor material.

Particularly preferred semiconductor nanocrystals for visible emission include CdX³, in which X³ is S, Se and Te, and ZnY³, in which Y³ It is Se, Te. For those molecules, ZnS is a preferred material for use as the coating. For CdTe, a preferred material may be ZnSe for use as the coating due to the greater degree of equality of crystal lattices between the materials. Coated nanocrystals that can be used in the present invention are described in Dabbousi et al. (1997) J. Phys. Chem. B, 101 (46) 9463, and Kuno et al., Supra.

Most of the prior art semiconductor nanocrystals are prepared in a coordination solvent, resulting in the formation of a passivating organic layer on the surface of the nanocrystal, composed of the organic solvent. Thus, passivated semiconductor nanocrystals are readily soluble in organic solvents, such as toluene, chloroform, and hexane. The present invention provides a surface modified particle that is soluble in aqueous media. According to the invention, the surface of the semiconductor nanocrystal is coated with an outer layer that stabilizes the semiconductor nanocrystal in aqueous solution. The outer layer includes a molecule that has at least one binding moiety that attaches it to the surface of the particle, and that ends in at least one hydrophilic moiety. Linker and hydrophilic moieties are optionally separated by a sufficient hydrophobic region to prevent charge transfer across the region. The hydrophobic region also provides a "pseudo-hydrophobic" environment for the nanocrystal, thereby protecting it from aqueous environments. In order for it to show high quantum efficiency, it is desirable that the particles remain electronically isolated from each other. The outer layer of the invention has such an additional useful purpose of maintaining the desired isolation between common semiconductor nanocrystals.

The outer layer can be made of any material that meets the structural and performance criteria set forth in this document. The material can be organic or inorganic. In preferred embodiments, the molecule is an organic molecule. In some embodiments, the outer layer can be a mixture of two or more different molecules that dissolve in water. In other embodiments, the outer layer may comprise additional molecules selected to provide a desirable attribute to the semiconductor nanocrystal. For example, the outer coating can include molecules that have functional groups reactive to react with other substances or molecules.

Suitable linking moieties include molecules that have electronic pairs available for interaction.

ES 2 228 107 T3 with the semiconductor surface, such as oxygen (O), sulfur (S), nitrogen (N) and phosphorus (P). Exemplary molecules include electron donating moieties, such as amines, thiols, phosphines, amine oxides, phosphin oxides, and the like. The linking moiety is attached to the surface of the semiconductor nanocrystal primarily through lone electronic pair coordination bonding of the nitrogen, sulfur, nitrogen or phosphorous atom of the linking group. Covalent bonding and ionic bonding can also be used to form the interaction of the outer layer with the surface of the semiconductor.

A molecule that exhibits a single binding moiety will result in the formation of an outer layer that exhibits water-solubilization properties; however, it may be desirable for the molecule to comprise a plurality of linker moieties, as schematically illustrated in Figure 2A. Thus, the molecule can be a bidentate or tridentate ligand having two or more linking groups 22, 22 '. Linking groups can be used as described herein above. For example, the molecule can be a dithiol, a diamine, a triamine, a diphosphine, and the like, derivatized. The linking groups can be the same or different.

Multidentate ligands provide improved stability and strength to the organic layer and the resulting water soluble nanocrystal. Without being tied to any particular mode of operation, it is believed that the improved stability of the water soluble nanocrystal is achieved by increasing the binding coefficient of the multidentate ligand to the surface of the semiconductor. Since the organic layer is formed by exchange reaction with solvated solvent molecules (see below), it is concluded that the molecule that solubilizes in water can also be displaced from the surface of the semiconductor nanocrystal. It has been observed, for example, that the outer layer can be at least partially removed by dialysis of the water-soluble layer. The use of a multidentate ligand increases the resistance of the interaction of the molecule with the semiconductor nanocrystal, and decreases the ease of exchange of the organic layer with other coordinating molecules.

An increase in the stability of the water-soluble semiconductor nanocrystal resulting in selective precipitation by sizes of coated semiconductor nanocrystals has been qualitatively observed. Semiconductor nanocrystals that have been coated with a bidentate ligand, such as lipoic acid, show a four-fold increase in suspension stability over a molecule coated with a comparable monodentate ligand.

The hydrophilic moiety can be a polar group or a charged group (positively or negatively). The polarity or charge of the group provides the necessary hydrophilic interactions with water to provide stable solutions or suspensions of the semiconductor nanocrystal. Exemplary hydrophilic groups include polar groups such as hydroxides (-OH), amines, polyethers such as polyethylene glycol, and the like, as well as charged groups, such as carboxylates (-CO₂^-), sulfonates (-SO3^-), phosphates (-PO4^-2) and phosphonates (-PO3^-2), nitrates, ammonium salts (-NH4⁺), and the like.

Water solubility has been achieved using molecules that have a single hydrophilic group; however, it may be desirable for the molecule to include more than a single hydrophilic moiety, as schematically illustrated in Figure 2B. Figure 2B shows a molecule that has at least two hydrophilic residues 24, 24 '. Hydrophilic groups can be the same or different. It is also contemplated that the water-solubilizing molecule may include multiple linking groups and hydrophilic groups, as shown in Figure 2C.

The hydrophobic region is selected to prevent photo-oxidation of the surface by transfer of charge from a hole to the surface, either from the core of the semiconductor nanocrystal or from the environment. Typical procedures include electrolysis of surrounding water with the resulting oxidation of sulfur or selenium (from the semiconductor nanocrystal) to SO₂ or SeO₂, in cases where the semiconductor nanocrystal or coating layer contains S or Se. Transfer of a charge through the layer represents a non-emissive energy pathway to the excited state of the semiconductor, thereby significantly reducing or quenching photoluminescence.

Prior art modifications for semiconductor nanocrystal surfaces include terminating CdS nanocrystals with 2-mercaptoethanol, 1-thioglycerol, and 3-mercaptopropionic acid. See, Lawless et al., Above, and Rogach et al., Above. These short chain organic molecules do not provide an optically luminescent water soluble semiconductor nanocrystal because the short carbon chain does not provide adequate isolation of the semiconductor nanocrystal from photooxidative processes. Therefore, charge transfer can occur between the semiconductor nanocrystal and the carboxylate or aqueous environment. Luminescence is partially extinguished, and quantum yields are low, ie less than 1%, in systems employing short-chain organic molecules as the termination layer.

In one embodiment of the invention, the hydrophobic region is a long chain hydrocarbon moiety, - (CH₂)_n-, where n is greater than six and preferably greater than eight. Hydrocarbon moieties where n equals 11 or 15 have been used successfully in the manufacture of the water-soluble nanocrystal of the invention. There is no upper limit to the length of the hydrocarbon chain. However, it is recognized that very long hydrocarbon chains can render the nanocrystal undesirably "greasy". The hydrophobic region can also include branched hydrocarbons.

In another embodiment, the hydrophobic region can include a modified hydrocarbon backbone. This modification can be the result of coupling reactions, eg, carbodiimide coupling, used to increase the length of the hydrophobic backbone. Alternatively, in the main chain you can enter

ES 2 228 107 T3 ducting non-carbon atoms, to enhance the attractive interaction of the water-solubilizing ligand with neighboring molecules.

The backbone can also be modified to include pendant groups that attract neighboring hydrophobic regions by forces such as van der Waals attraction or by hydrogen bonding. The attractive interaction between neighboring molecules serves to stabilize the outer layer of the semiconductor nanocrystal. In the case where the bonding moiety dissociates from the surface of the semiconductor, the attractive interaction with its neighbors will help the molecule to remain closely associated with the semiconductor nanocrystal until its bonding moiety is able to re-coordinate itself to the surface.

Exemplary modifications include amide, ketone, ether, and aromatic moieties, and the like, which replace all or part of the hydrocarbon backbone, or which are attached as pendant groups from the hydrocarbon chain. The polar nature of the residues promotes hydrogen bonding and other attractive interactions with neighboring molecules, stabilizing the coating and increasing its strength in aqueous solution.

In other embodiments of the invention, the outer layer molecule is cross-linked or polymerized with its neighboring molecules. Crosslinking provides stability to the layer by creating an effectively multidentate ligand across the entire surface of the semiconductor, and significantly reducing the volatility of the ligand and increasing the strength and stability of the coating. In Figure 3, exemplary cross-linked networks are schematically illustrated.

To this end, the hydrocarbon chain can include some degree of unsaturation, which can crosslink upon exposure to UV energy or other free radical initiator, to form bridges between neighboring ligands. The unsaturation of the hydrocarbon (and subsequent crosslinks) retains the desired hydrophobicity to avoid photoinduced degradation of the semiconductor surface.

In one embodiment of the invention, the outer layer ends in an unsaturated hydrophilic moiety that is capable of cross-linking or polymerization. For example, the unsaturated moiety can be an acrylate or methacrylate, which can be polymerized by exposure to free radical initiators, heat, UV energy, etc., to form poly (methacrylate), as shown in Figure 4. The result is a polymeric network, in this example poly (methacrylate), that interacts with, and effectively protects, the semiconductor nanocrystal from an aqueous environment. The poly (methacrylate) can be deprotonated to provide a charged surface and make the nanocrystal water soluble. Other exemplary unsaturated moieties for polymerization include acrylic acid and polystyrene derivatized to include a water-solubilizing functional group, eg, carboxylate and sulfonate, and the like.

In another embodiment of the invention, the outer layer comprises a block copolymer that provides the required binding, hydrophilic and hydrophobic functionalities. The copolymer includes at least a first block containing a pendant group capable of functioning as a linking moiety, and a second block having a pendant group capable of functioning as a hydrophilic moiety. The polymeric backbone can function as the hydrophobic region. The linking and hydrophilic moieties can be attached directly to the hydrocarbon backbone, or they can be attached via intermediate spacer groups. For example, the linking group Y may terminate from an aromatic or alkyl spacer group to provide greater access to the semiconductor surface.

In one embodiment of the invention, the molecule has the structural formula (V):

--Y²(R¹) -— - R² I X '(R⁴) |. (V) in which R¹, R², R⁴, X², Y², m 'and n' are as defined above. In an exemplary embodiment of a molecule having the structural formula (V), the molecule is a block copolymer having the formula

<img file="ES2228107T3_D0004.tif" />

wherein X and Y are linking moieties and hydrophilic moieties, respectively, and can be any of the moieties discussed hereinbefore. R and R 'can be hydrogen, R can be a polar residue, and R' can be a non-polar residue. The block copolymer can have a molecular weight between 300 and 50,000. The block sizes for the hydrophilic and linker moieties are preferably in the range of about 3 to 100.

Exemplary molecules for use in the invention have the structural formula (I)

ES 2 228 107 T3 (I)

H_zX ((CH2)_nCÜ2H)_Y where X, z, n and y are as defined above, the structural formula (II)

<img file="ES2228107T3_D0005.tif" />

(II) or structural formula III

<img file="ES2228107T3_D0006.tif" />

where Y, Z, X, X 'and X "are as defined above, or structural formula (IV) (R¹)_to - R² - [(R³) b (R⁴) c] d (IV) in which R¹, R², R³, R⁴, a, b, c and d are as defined above.

Exemplary molecules for use in the outer layer of the water soluble nanocrystal of the invention having the formula provided herein above include long chain aminocarboxylic acids, NH₂(CH₂)_nCOOH, and phosphinecarboxylic acids P ((CH₂)_nCOOH)₃, and its oxides O = P ((CH₂)_nCOOH)₃, where n is greater than or equal to 6, preferably n is greater than or equal to 8, and more preferably n is equal to 10 to 12. The carboxylic acid can be deprotonated to provide the hydrophilic moiety. Other suitable molecules include bidentate ligands, such as, dihydrolipoic acid, HSCH₂CH₂CH (SH) (CH₂)₄COOH, or more generally, HSCH₂CH₂CH (SH) (CH₂)_nCOOH, where n equals 1 to 10. The length of the ligand can be increased by standard carbodiimide coupling procedures, yielding a species with the formula HSCH₂CH₂CH (SH) (CH₂)₄CONH (CH₂)_nCOOH. The commercial availability of numerous precursors allows n to easily range from 2 to at least 10. In Rich et al. (1979) The Peptides Vol. 1, Academic Press, pp. 241-2561 more details of the carbodiimide coupling reaction can be found.

Other suitable bidentate ligands include: the primary amine-containing analogs of the above molecule, H₂NCH₂CH₂CH (NH₂) (CH₂)_nCOOH; derivatives of ethylenediamine, such as (HOOC (CH₂)_n) HNCH₂CH₂NH ((CH₂)_nCOOH); diphosphines such as (HOOC (CH₂)_n)₂PCH₂CH₂P ((CH₂)_nCOOH)₂; and the corresponding diphosphine oxides (HOOC (CH₂)_n)₂P (O) CH₂CH₂P (O) ((CH₂)_nCOOH)₂. An advantage of using the carboxylic acid derivatives mentioned above is that they themselves lend themselves to a wide range of chemistries. For example, the water soluble semiconductor nanocrystal can be coupled with molecules that have biological affinity for use in assays. In another example, the water soluble semiconductor nanocrystal can be attached to beads, solid supports, or objects of interest in order to track or identify an item. See US Patents n^you 6,426,513 and 6,617,583, above, for additional details.

It will be readily apparent to one of ordinary skill in the art that the carboxylic acid moiety of the molecules listed above can be substituted with a wide variety of charged or polar groups, including but not limited to hydroxides, polyethers, such as polyethylene glycol and the like, and amines, as well as charged groups such as carboxylates, sulfonates, phosphates, nitrates, ammonium salts, and the like. Molecules such as those listed hereinabove are commercially available or can be synthesized from procedures and procedures well known in the art. Additionally it will be apparent that the modifications described above with respect to hydrophobic regions and hydrophilic groups can be incorporated into the molecule described immediately above in the preparation of suitable ligands for use in the outer coating of the invention.

In another aspect of the invention, the water soluble outer layer may be a bilayer comprising a layer

Inner ES 2 228 107 T3, which has an affinity for the semiconductor surface, and an outer layer, which ends in a hydrophilic layer that has an affinity for an aqueous medium. Figure 5A illustrates an exemplary molecule used in the outer bilayer of the invention. The molecule, dioctyl sulfosuccinate (OT ™ aerosol), contains hydrophobic hydrocarbon regions 52 (represented schematically as "-----" in Figure 5A), and a charged hydrophilic region 54 (represented by "O" in Figure 5A). An exemplary bilayer molecule is shown in Figure 5B, in which an inner layer 40 includes a molecule 42 (here TOPO) having a binding moiety 44 with an affinity for the surface of the semiconductor. A hydrophobic tail 48 extends from the linking moiety. The second outer layer 50 comprises an inner hydrophobic region 52 and a terminal hydrophilic moiety 54 for favorable interaction with an aqueous medium. The hydrophobic regions 48, 52 of the inner and outer layers, respectively, interact preferentially in the aqueous medium, to form a micelle that encapsulates the nanocrystal therein. Figure 5B also illustrates the displacement reaction that occurs to form the bilayer of the invention.

The inner layer can include those coordination solvents typically used in the manufacture of the semiconductor nanocrystal. Exemplary molecules include trialkylphosphines and phosphine oxides, such as trioctylphosphine oxide (TOPO), trioctylphosphine (TOP), tributylphosphine (TBP), and the like. One possible solvent is hexadecylamine, in particular to dissolve ZnSe.

The second outer layer can include any surfactant that has a non-polar tail and a polar head. Non-limiting examples of surfactants include sodium dioctylsulfosuccinate (known by the trade name AOT soap), C₁₂(OC ^ C ^^ OH (Brij 35®), C1₈H3₇(OCH2CH2) 1qOH (Brij 76®) and C ^ (OC ^ C ^ qOH (Brij 98®). Even the usual hand soap, for example Ivory® soap, has been used successfully in the preparation of nanocrystals of the invention soluble in water.

A procedure for the preparation of the water soluble nanocrystal is as follows. The procedure is described for a CdSe (ZnS) nanocrystal, that is, a CdSe core with a ZnS shell, but it is understood that the procedure can be applied in the preparation of semiconductor nanocrystals from known semiconductor materials.

A population of nearly monodisperse nanocrystals is prepared. The actual size of the nanocrystals will vary depending on the material used. For CdSe, the particles range in size from about 12 A to about 150 A in diameter, with a particle size distribution of about 5 to 10% rms in diameter. Monodisperse nanocrystals can be obtained using a high temperature colloidal growth procedure, optionally followed by size selective precipitation. If the spectral emission line widths are not as narrow as desired, size selective precipitation can be used to obtain a population of semiconductor nanocrystals of a narrower particle size distribution. See, Murray et al. (1993), above, the thesis of Murray (1995), above, and Kuno et al., Above.

The core of the semiconductor nanocrystal can then be coated with the appropriate coating layer of the semiconductor, ie the shell. The coated nanocrystal can be prepared by introducing the first substantially monodisperse semiconductor nanocrystal and a precursor capable of thermal conversion to a second semiconductor material in a coordination solvent. The coordination solvent is maintained at a temperature sufficient to convert the precursor to the second semiconductor material, but insufficient to substantially alter the monodispersity of the first semiconductor nanocrystal. Preferably, the second semiconductor material has a higher bandgap than that of the first semiconductor nanocrystal. A cladding shell of the second semiconductor material is formed on the first semiconductor nanocrystal. The monodispersity of the nanocrystal is monitored during the conversion of the precursor and coating of the first semiconductor nanocrystal. The particle size distribution can be further refined by size selection precipitation. More details on preparing a nanocrystal can be found in US Patent No. 6,322,901, filed November 13, 1997, and entitled "Highly Luminescent Color-Selective Materials" and Dabbousi et al., Above. coated semiconductor for use in the water soluble nanocrystal of the invention.

The outer surface of the nanocrystal, as formed, includes an organic layer derived from the coordination solvent used during the termination layer growth process. The surface of the nanocrystal can be modified to obtain the nanocrystal of the invention soluble in water, by repeated exposure to an excess of a competing coordinating group. For example, a dispersion of the semiconductor nanocrystal can be treated with a coordinating organic molecule, such as those described herein, to produce nanocrystals that are easily dispersed in water, but no longer dispersed in aliphatic compounds. Such a surface exchange procedure can be carried out using a variety of molecules that are capable of coordinating or bonding to the outer surface of the plugged semiconductor nanocrystal, such as, by way of example, phosphines, thiols, amines, phosphine oxides and amine oxides.

A typical reaction is illustrated in Figure 6. The semiconductor nanocrystals 60 are prepared in a coordinating organic solvent, such as trioctylphosphine oxide (TOPO), which results in the formation of a passivating TOPO layer 62 on the surface of the semiconductor nanocrystal. This layer is displaced, at least in part, by ligand 54, represented here as a long-chain mercaptocarboxylic acid, comprising the outer layer of the invention, in order to obtain the water-soluble nanocrystal 66. Displacement can occur by dispersion of the semiconductor nanocrystals or coated semiconductor nanocrystals

ES 2 228 107 T3 in a medium containing high concentrations of the ligand used to form the outer coating. The medium can be a neat liquid comprising the ligand, or it can be a highly concentrated solution. High concentrations cause the displacement reaction to take place to maximize coverage of the nanocrystal surface by the outer coating molecule. Note that the TOPO layer displacement does not need to be complete in order to obtain a water soluble nanocrystal.

Repeated exposure of the nanocrystal to the coordinating ligand solution may be desirable. The outer coating may comprise a mixture of the original polar organic solvent used in the preparation of the nanocrystal and the water-solubilizing molecule used in the outer coating of the invention. The substitution of the water-solubilizing molecule may be sufficient to render the molecule water-soluble, and need not be complete. In some embodiments, the substitution is about 25 to 50% complete, preferably 60% complete. The actual degree of substitution necessary for water solubility will depend on the number of charged or polar groups on the water solubilizing molecule. A greater number of charged or polar groups may require a lower level of surface substitution in order to achieve water solubility.

It is also within the scope of the present invention to include other coordinating ligands in the outer coating of the nanocrystal. Additional ligands can be included so that additional chemical reactions can be performed to the nanocrystal. For example, coordinating ligands terminating in reactive groups such as carboxylic acid, acyl halides, and the like can be added to the outer surface of the nanocrystal.

It should be understood that, although the invention has been described in conjunction with specific preferred embodiments thereof, the description as well as the examples that follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention belongs.

The following examples are intended to provide those skilled in the art with a complete description and discussion of how to obtain and use the novel compositions of the invention, and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure accuracy with respect to the numbers used (eg quantities, temperatures, etc.), but of course some errors and experimental deviations must be allowed. Unless otherwise indicated, parts are parts by weight, temperatures are in degrees centigrade, and pressure is at or near atmospheric.

The practice of the present invention will employ, except where otherwise indicated, conventional techniques of synthetic organic chemistry, biochemistry, molecular biology, and the like, which are within the skill of the art. These techniques are fully explained in the literature. See, for example, Kirk-Othmer's Encyclopedia of Chemical Technology; House's Modern Synthetic Reactions; the text by Marvel et al. ORGANlC SYNTHESIS; Volume 1 of the collection, and the like.

Example 1

Preparation of TOPO-terminated CdSe (ZnS) (a) Preparation of CdSe. Trioctylphosphine oxide (TOPO, 90% purity) and trioctylphosphine (TOP, 95% purity) were obtained from Strem and Fluka, respectively. Dimethylcadmium (CdMe₂) and diethylzinc (ZnEt₂) were purchased from Alfa and Fluka, respectively, and both materials were filtered separately through a 0.2 m filter in an inert atmosphere box. Trioctylphosphine selenide was prepared by dissolving 0.1 mole of Se pellets in 100 ml of TOP thereby producing a 1M solution of TOPSe. Hexamethyl (disilatiano) (TMS2S) was used as purchased from Aldrich. HPLC grade n-hexane, methanol, pyridine, and n-butanol were purchased from EM Sciences.

The typical preparation of TOP / TOPO-terminated CdSe nanocrystals is as follows. TOPO (30 g) was placed in a flask, and dried under vacuum (~ 1 Torr) at 180 ° C for 1 hour. The flask was then filled with nitrogen and heated to 350 ° C. The following injection solution was prepared in a dry box under an inert atmosphere: CdMe₂(200 microliters, 2.78 mmol), 1 M TOPSe solution (4.0 ml, 4.0 mmol), and TOP (16 ml). The injection solution was mixed thoroughly, loaded into a syringe, and removed from the dry box.

The heat was removed from the reaction flask, and the reagent mixture was delivered with a single continuous injection into the vigorously stirred TOPO. This produces a deep yellow / orange solution, with a marked absorption characteristic between 470 and 500 nm and a sudden drop in temperature to ~ 240 ° C. Heating was restored to the reaction flask, and the temperature was gradually raised to 260-280 ° C.

Aliquots of the reaction solution were withdrawn at regular intervals (5-10 minutes), and absorption spectra were taken to monitor the growth of crystallites. The best samples were prepared during a period of constant growth of a few hours by modulating the growth temperature in response to changes in the size distribution, as estimated from the graph in the absorption spectra. The temperature decreased between 5 and 10 ° C in response to an increase in the size distribution. Alternatively, the reaction can also be stopped at this point. When growth seems to stop, the temperature rises by 5-10 ° C. When the desired absorption characteristics were observed, the reaction flask was allowed to cool to about 60 ° C, and 20 ml of butanol was added to avoid solidification of the TOPO. Adding a high excess of methanol

ES 2 228 107 T3 causes the particles to flocculate. The flocculate was separated from the supernatant liquid by centrifugation. The resulting powder can be dispersed in a variety of organic solvents (alkanes, ethers, chloroform, tetrahydrofuran, toluene, etc.) to produce an optically clear solution.

Additionally the powder can be optimized in an optional precipitation procedure by size selection. The nanocrystallites were dispersed in a solution of ~ 10% butanol in hexane. Then, methanol was added dropwise to this stirred solution until opalescence persisted. Separation of the supernatant and flocculate by centrifugation produced a precipitate enriched with the largest crystallites in the sample. This procedure was repeated until no more sharp jumps in the optical absorption spectrum were observed. Size selection precipitation can be carried out in a variety of solvent / non-solvent pairs, including pyridine / hexane and chloroform / methanol.

(b) Preparation of CdSe (ZnS). A flask containing 5 g of TOPO was heated to 190 ° C under vacuum for several hours, then cooled to 60 ° C after which 0.5 ml of trioctylphosphine (TOP) was added. Approximately 0.1 to 0.4 micromoles of CdSe nanocrystals dispersed in hexane were transferred into the reaction vessel via syringe, and the solvent was pumped off.

As the precursors of Zn and S, diethylzinc (ZnEt₂) and hexamethyldisilatiano ((TMS)₂S). The particle size distribution for a particular sample was determined by comparing the optical data with that of known semiconductor nanocrystals of known particle size. The amounts of Zn and S precursors needed to grow a ZnS shell of the desired thickness for each CdSe sample was calculated based on the ratio of shell volume to core volume, assuming spherical core and shell, and taking into account the mass network parameters of CdSe and ZnS. For larger particles, the ratio of Zn to Cd required to achieve the same thickness crust is lower than for smaller nanocrystals. The actual amount of ZnS growing on the CdSe nuclei was generally less than the amount added due to incomplete reaction of the precursors and the loss of some of the material on the flask walls during the addition.

Equimolar amounts of the precursors were dissolved in 2 to 4 ml of TOP inside a glove box with an inert atmosphere. The precursor solution was loaded into a syringe and transferred to an addition funnel attached to the reaction flask. The reaction flask, containing the CdSe nanocrystals dispersed in TOPO and TOP, was heated in an atmosphere of N2. The temperature at which the precursors were heated ranged from 140 ° C for 23A diameter nanocrystals to 220 ° C for 55 A diameter nanocrystals. When the desired temperature was reached, the Zn and S precursors were added dropwise to the vigorously stirred reaction mixture, over a period of 5 to 10 minutes.

After the addition was complete, the mixture was cooled to 90 ° C and allowed to stir for several hours. Butanol (5 ml) was added to the mixture to prevent the TOPO from solidifying on cooling to room temperature. The coated particles were stored in their growth solution to ensure that the surface of the nanocrystals remained passivated with TOPO. They were then recovered as a powder by precipitating with methanol and redispersing in a variety of solvents including hexane, chloroform, toluene, THF, and pyridine.

Example 2

Preparation of water soluble nanocrystals, using long chain mercaptocarboxylic acid

The TOPO-terminated CdSe (ZnS) semiconductor nanocrystals were prepared as described in Example 1. The coated CdSe (ZnS) nanocrystals were precipitated from the growth solution using a mixture of butanol and methanol. To obtain the precipitated semiconductor nanocrystals, the solution was centrifuged for 5 to 10 minutes, the supernatant was decanted, and the residue was washed with methanol (2X).

The residue was weighed. The weight of the TOPO finish was assumed to be 30% of the total weight; and a 30-fold molar excess of the new terminator molecule, 11-mercaptoundecanoic acid (MUA), was added. The residue and MUA (neat solution) were stirred at 60 ° C for 8 to 12 hours. While the mixture was still warm, a volume of tetrahydrofuran (THF) equal to the added MUA was added to the MUA / nanocrystal mixture. A clear solution resulted, and the coated semiconductor nanocrystals were stored in THF.

The coated semiconductor nanocrystals are made water soluble by deprotonation of the carboxylic acid functional group of the MUA. Deprotonation was achieved by adding a suspension of potassium t-butoxide in THF to the semiconductor nanocrystal-MUA / THF solution. A gel was produced which was then centrifuged, and the supernatant was poured off. The residue was washed twice with THF, centrifuged each time, and the supernatant was poured off. The final residue was allowed to air dry for 10 minutes. Deionized water (Millipore) was added to the residue until a clear solution formed.

The resulting coated semiconductor nanocrystals were analyzed for photoluminescent quantum yield. A CdSe semiconductor nanocrystal, with a ZnS four monolayer coating coated as described, had an absorption band at 480 nm and a photoluminescent band at 500 nm, with a quantum yield of 12%. A second semiconductor nanocrystal of CdSe, with a coating of four ZnS monolayers

ES 2 228 107 T3 coated as described, had an absorption band at 526 nm and a photoluminescent band at 542 nm, with a quantum yield of 18%.

Example 3

Preparation of a water-soluble semiconductor nanocrystal, using a multidentate ligand

A water soluble semiconductor nanocrystal was prepared as described in Example 2, except that the bidentate ligand dihydrolipoic acid was used.

The synthesis of a dithiolic bidentate ligand was achieved by reducing the coenzyme lipoic acid. The general procedure is described in Gunsalus et al. (1956) J. Am. Chem. Soc. 78: 1763-1766. Sodium borohydride (1.2 g) was added in 30 to 50 mg portions to a stirred suspension of lipoic acid (6.0 g) in 117 ml of 0.25 M sodium bicarbonate in water at 0 ° C. The reaction was stirred for 45 minutes at 0 ° C, then 100 ml of toluene was added and the mixture was acidified to pH ~ 2 with hydrochloric acid. The toluene layer was collected and stored. The aqueous layer was washed three times with 15 ml of toluene. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed in vacuo, leaving the dihydrolipoic acid product as a yellow oil (80% yield).

Terminator molecule exchange was performed using the same procedure as described for 11-mercaptoundecanoic acid. The TOPO-terminated CdSe (ZnS) semiconductor nanocrystals were precipitated from solution and washed twice with methanol. The remaining powder was dissolved (under nitrogen) at 70 ° C in the minimum amount (usually between 300 and 600 mg) of dihydrolipoic acid necessary to produce a clear solution. This mixture was stirred at 70 ° C for 6 hours, and then stored at room temperature. The nanocrystals were made water soluble by treatment with potassium t-butoxide in THF, as described for mercaptocarboxylic acid ligands.

Example 4

Preparation of a water-soluble semiconductor nanocrystal, using a surfactant

The TOPO-terminated CdSe (ZnS) semiconductor nanocrystals were prepared as described in Example 1. The semiconductor nanocrystals were dissolved in hexane to give a solution having a concentration of between approximately 0.001 and 0.01 molar CdSe nanocrystals. (ZnS). Sufficient sodium dioctylsulfosuccinate surfactant (trade name AOT) was added to the mixture to produce a solution having 5% surfactant by weight (but Ivory® liquid soap will also work). The hexane solvent was evaporated in vacuo. The resulting solid residue was dissolved in water to give a clear solution whose quantum yield was approximately the same as the initial sample (~ 75% of the original value).