WO2008140495A2

WO2008140495A2 - A method to produce water-dispersible highly luminescent quantum dots for biomedical imaging

Info

Publication number: WO2008140495A2
Application number: PCT/US2007/024321
Authority: WO
Inventors: Ken-Tye Yong; Indrajit Roy; Paras N. Prasad
Original assignee: Research Foundation of the State University of New York
Current assignee: Research Foundation of the State University of New York
Priority date: 2006-11-22
Filing date: 2007-11-21
Publication date: 2008-11-20
Anticipated expiration: 2009-05-22
Also published as: WO2008140495A3

Abstract

The invention disclosed herein provides a quick, low-temperature method for production of highly luminescent, CdS/ZnS graded-shell passivated, CdSe-core-nanocrystal quantum dots. The method uses non-toxic, easy-to-handle cadmium, zinc and sulfur precursors. Quantum dots produced using the present method are suitable for bioimaging applications.

Description

AMETHOD TO PRODUCE WATER-DISPERSIBLE HIGHLY LUMINESCENT QUANTUM DOTS FOR BIOMEDICAL IMAGING

This application claims priority to U.S. Provisional Application No. 60/866,984 filed on November 22, 2006, the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the field of nanoscale materials preparation and more particularly provides an efficient, low-temperature preparation of II- VI semiconductor quantum dots using non-toxic, conveniently handled precursor materials.

BACKGROUND OF THE INVENTION

Semiconductor quantum dots (QDs) are slowly replacing molecular fluorophores as optical contrast agents for bioimaging applications by virtue of their significantly improved spectral stability as well as their tunable optical properties (e.g. emission wavelength and absorption wavelength). Other advantages of QDs include broad and continuous absorption with high molar extinction coefficients, a narrow and symmetric emission, often with high photoluminescence quantum yield (QY), as well as a strong resistance to photobleaching. Ever since the first reports of their use as biomarkers several years ago, a wide variety of biological applications have been demonstrated, including those with emission in the near infrared (NIR) range. The biological applications of QDs include robust tumor targeting in vitro and in vivo, long-term in vivo observation of cell-trafficking and study of intracellular events within the resolution of a single molecule, to name a few. The main reason for such versatility is their rich surface chemistry in the aqueous phase, which makes it possible to incorporate a wide spectrum of biomolecules (protein, peptide, DNA, etc.) designated for performing a specific function. Currently, CdS/ZnS graded-shell passivated CdSe (CdSe/CdS/ZnS) QDs are generally prepared using a hot colloidal method which requires high reaction temperatures and toxic, diffϊcult-to-handle and expensive reagents. For example, the conventional method for producing CdSe/CdS/ZnS QDs generally uses toxic starting materials which need special care due to volatility and/or reactivity or other similar properties. Typically, an inert- atmosphere glove box, which is cumbersome to use, is needed to prepare and store conventionally used dimethyl cadmium, dimethyl zinc and hexamethyldisilathiane precursors because they are extremely toxic, pyrophoric and unstable at room temperature (these precursors are explosive and release large amounts of gas at elevated temperatures). Another conventionally used sulfur precursor is hydrogen sulfide gas (H₂S) which is also toxic and difficult to handle.

The toxicity and high cost of reagents, and the complexity of the reactions poses challenges for the manufacture of quantum dots. However, because of the potential for use of QDs in a wide range of biological applications, there exists an ongoing need for development of efficient and safe synthetic methods to produce QDs for use.

SUMMARY OF THE INVENTION

The present method is a low-temperature method for producing highly luminescent CdSe/CdS/ZnS quantum dots (QDs). Unlike the conventional, hot colloidal method the present method uses precursors which are non-toxic and do not require special handling (e.g. an inert atmosphere glove box is not required). The QDs produced using the method described herein are easily rendered dispersible in aqueous solution. The QDs are suitable for bioimaging applications.

The method is comprised of the following steps: reaction of a cadmium precursor and trialkylphosphine-selenium solution to form CdSe-core nanocrystals; reaction of the

CdSe-core nanocrystals, a cadmium precursor, a zinc precursor and a trialkylphosphine-sulfur solution to form CdS/ZnS graded-shell passivated, CdSe-core nanocrystal quantum dots (CdSe/CdS/ZnS QDs); reacting the CdSe/CdS/ZnS QDs with a mercapto-containing ligand; and deprotonating the mercapto-containing ligand. The result is production of aqueous- dispersible CdSe/CdS/ZnS QDs.

In one embodiment cadmium oxide and a trioctylphosphine-selenium solution are reacted to form CdSe-core nanocrystals. The CdSe-core nanocrystals are combined with cadmium oxide and zinc acetate, and a trioctylphosphine-sulfur solution is subsequently added. The resulting organic-soluble CdSe/CdS/ZnS QDs are combined with mercaptosuccinic acid. The resulting mercaptosuccinic acid-associated, CdSe/CdS/ZnS QDs are deprotonated with ammonium hydroxide.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. TEM (transmission electron microscopy) image of as-synthesized, organic-soluble CdSe/CdS/ZnS QDs. The size of the QDs is estimated to be 6.5 nm. This value was estimated by averaging the size of 150 particles. The scale bar is 100 nm.

Figure 2. Powder XRD (x-ray diffraction) profile from CdSe/CdS/ZnS quantum dots. All the diffraction peaks can be readily indexed to the wurtzite structure of CdSe. The intensity of the (002) diffraction peak is much stronger than that of all other peaks, suggesting that the CdSe/CdS/ZnS QDs have a strong preferential orientation along the [001] direction. These signals suggest that the particles have well developed crystallinity.

Figure 3. EDS (energy dispersive spectroscopy) spectrum of CdSe/CdS/ZnS quantum dots. From the EDS analysis, it is evident that the only elements present in the QDs sample are cadmium, selenium, sulfur, and zinc.

Figure 4. Three batches of as-synthesized, organic-soluble CdSe/CdS/ZnS QDs produced using the protocol described in Example 1. The emission peak varied from 608 to 630 nm. The highest quantum yield observed was ~60% and the lowest ~20%. Figure 5. Confocal image of cancer cells labeled with aqueous-dispersible CdSe/CdS/ZnS QDs.

Figure 6. TEM image of water-dispersible CdSe/CdS/ZnS QDs.

Figure 7. Tunable emission spectra between 470 nm and 600 run from different sizes of CdSe-core nanocrystals.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a quick, low-temperature method for the preparation of highly luminescent CdS/ZnS graded-shell passivated, CdSe QDs using non- toxic starting materials which do not require special handling methods. Gram-scale quantities of CdSe- based QDs with a quantum yield as high as 60% and tunable optical properties can be produced using the procedure disclosed herein, hi one embodiment aqueous-dispersible CdSe/CdS/ZnS QDs are produced using this method. In another embodiment aqueous- dispersible CdSe/CdS/ZnS QDs with an average diameter of 6.5 nm are produced, hi yet another embodiment aqueous-dispersible CdSe/CdS/ZnS QDs with an emission wavelength of 600 to 660 nm are produced, hi yet another embodiment, the emission wavelength of the QDs is 630 to 650 nm.

The present method addresses disadvantages associated with the conventional method. Unlike the precursors used in the conventional method, the precursors used in the present method are non-toxic and can be handled without any special precautions, e.g. use of an inert atmosphere glove box is not required. The use of precursors which are toxic and require special handling precautions becomes especially problematic as the reaction is scaled to a commercially practicable level. Use of the precursors described herein becomes increasingly valuable as the scale of the reaction increases.

Also, unlike the conventional method, completion of the present method does not require long periods of time. The entire process is fast and can be completed within 3-8 hours while the conventional method requires up to 24 hours to complete.

Furthermore, the present method requires lower reaction temperatures than the conventional method. For example, the CdSe-core nanocrystal reaction can be run at temperatures as low as 180⁰C. In comparison, preparation of CdSe-core nanocrystals and growing a CdS/ZnS graded shell using the conventional method requires reaction temperatures in excess of 320°C.

To maximize the quantum yield of CdSe QDs (an important property for bioimaging applications) the surface of CdSe-core nanocrystals is preferably passivated to minimize non- radiative relaxation pathways (e.g. surface defects which are referred to as traps) which result in a decrease in quantum yield. Passivation can be accomplished by growing a shell layer of a wider-band gap semiconductor material. Zinc sulfide (ZnS) has been used in this capacity. In addition, ZnS also prevents oxidation of the CdSe-core nanocrystal. Oxidation can lead to leaching of harmful cadmium and selenium compounds making ZnS-passivated CdSe QDs suitable materials for bioimaging applications.

The problem with ZnS passivated CdSe QDs is that the ZnS crystal lattice parameters are a poor match (-12%) with those of CdSe. This lattice mismatch results in strain at the interface between the CdSe-core nanocrystal and ZnS shell. When the ZnS shell exceeds two monolayers the interfacial strain can lead to defects in the ZnS shell which negatively impact their photoluminescence efficiency and stability, and their colloidal stability.

This interfacial strain problem can be alleviated by growing a graded CdS/ZnS shell to passivate the CdSe-core nanocrystal. The lattice parameters of CdS are a better match with those of CdSe than those of ZnS. In the present method, this better match of lattice parameters leads to preferential growth of a CdS layer on the CdSe-core nanocrystal. The CdS layer mediates growth of the final outer ZnS shell. The present method results in growth of a graded CdS/ZnS passivation layer. The passivation layer is primarily CdS at the CdSe-core nanocrystal-passivation layer interface and the Zn incorporation increases, and Cd incorporation correspondingly decreases, as the passivation layer grows. Growth of a graded CdS/ZnS passivation layer results in improved aqueous stability and improved quantum yield. For example, CdSe/ZnS QDs have a quantum yield of only 3% whereas CdSe/CdS/ZnS QDs have a quantum yield above 20%.

The present method is comprised of the following steps. First, CdSe-core nanocrystals are prepared. Second, a CdS/ZnS graded shell is grown on the CdSe-core nanocrystals yielding CdSe/CdS/ZnS QDs. Third, CdSe/CdS/ZnS QDs are reacted with mercapto-group containing ligands and the ligand-associated CdSe/CdS/ZnS QDs are deprotonated to produce aqueous-dispersible CdSe/CdS/ZnS QDs.

In the first step, preparation of the CdSe core nanocrystals, a cadmium precursor (0.1 to 30 mmol) is dissolved in a solvent system comprised of a coordinating solvent and a surfactant. In one embodiment examples of suitable cadmium precursors are cadmium oxide, cadmium chloride, cadmium acetate, cadmium acetylacetonate, and cadmium nitrate. This mixture is heated slowly under an argon atmosphere to a temperature between 18O⁰C and 300⁰C. In one embodiment the reaction temperature is 275°C to 285°C with an average temperature of 280⁰C. Examples of suitable coordinating solvents are C₁₃ to C₂i, saturated and unsaturated fatty acids (e.g. oleic acid, myristic acid, palmitic acid, stearic acid, arachidic acid, linoleic acid, erucic acid, and behenic acid). Examples of suitable surfactants are C₆ to Ci₈ alkylphosphonic acids (e.g n-tetradecylphosphonic acid, n-decylphosphonic acid, n- dodecylphosphonic acid, n-hexadecylphosphonic acid, n-hexylphosphonic acid, n- octadecylphosphonic acid, and n-octylphosphonic acid).

A trialkylphosphine solution of selenium is rapidly injected into the reaction mixture. Examples of suitable trialkylphosphines include trioctylphosphine and tributylphosphine. An unsaturated monoalkylamine can be substituted for the trialkylphosphine. An example of a suitable monoalkylamine is oleyamine. Aliquots (0.1 to 10 mL) are withdrawn from the reaction mixture. For example, aliquots may be withdrawn from the reaction mixture from 5 seconds to 10 min after injection of the selenium solution. The aliquot is quenched in chloroform and CdSe-nanocrystals separated from the surfactant solution by addition of ethanol and centrifugation within the range of 3,000-15,000 rpm.

The size of the CdSe-core nanocrystals, and hence their emission wavelength, is controlled by how long the reaction is allowed to proceed. The size is monitored by determining the emission profile of the nanocrystals isolated from each aliquot. When the desired emission profile is obtained the reaction is quenched with chloroform and the nanocrystals isolated by addition of ethanol and centrifugation. Figure 7 shows emission spectra for different sizes of CdSe-core nanocrystals.

In the second step, growth of a graded CdS/ZnS shell on the CdSe-core nanocrystals, a solution of CdSe-core nanocrystals (0.1 to 1 g) in hexane is prepared. Separately, a cadmium precursor (0.1 - 6 mmol) and a zinc precursor (0.1 - 6 mmol) are dissolved in a coordinating solvent (5-20 mL) and a trialkylphosphine oxide (1-20 g) to form a precursor solution, hi one embodiment examples of suitable zinc precursors are zinc acetate, zinc oxide, zinc chloride, zinc acetylacetonate, zinc nitrate, zinc undecylanate, and zinc 2- ethylhexanoate. An example of a suitable trialkylphosphine oxide is trioctylphosphine oxide (TOPO). hi one embodiment, the cadmium to zinc molar ratio is 1 : 1 to 1.8. The reaction solution is heated to a temperature of 100°C to 220°C and held at temperature for 45 minutes under an argon flow, hi one embodiment the temperature is from 150°C -190°C. CdSe-core-nanocrystals solution is injected slowly into the hot reaction mixture while the reaction mixture is stirred. The reaction mixture is held at a temperature of 150- 180°C with a needle outlet allowing the hexane to evaporate. After holding the reaction mixture at temperature for approximately 2 to 30 minutes, the needle is removed, and the reaction mixture heated to a temperature of 150⁰C to 28O⁰C. hi one embodiment the temperature is

235°C to 245°C and the average temperature is 240⁰C. Upon reaching temperature, a solution of elemental sulfur (1-2 mmol) in a trialkylphosphine (0.1 to 5 mL) is added drop wise to the reaction mixture with stirring. An example of a suitable trialkylphosphine is trioctylphosphine (TOP). The reaction mixture was held at temperature for 15 to 20 minutes. In one embodiment the temperature is between 235°C and 245°C and the average temperature is 240°C. The thickness of the shell is dependent on the concentration of the sulfur-TOP solution added into the reaction mixture. The shell thickness was determined by comparing the size of the CdSe-core nanocrystal and CdSe/CdS/ZnS QDs as determined by transmission electron microscopy. In one embodiment, the thickness of the shell is 1-2.5 nm. Once the desired shell thickness is achieved, the reaction mixture is quenched by large volume of organic solvent (5-50 mL), e.g. chloroform, hexane, and toluene. The CdSe/CdS/ZnS QDs are separated from the extra surfactant solution by addition of ethanol and centrifugation (11,000-15,000 rpm). In one embodiment the volume ratio of ethanol to nanocrystals solution is from 40:60 to 80:20. The precipitated CdSe/CdS/ZnS QDs can be readily be dispersed in organic solvents such as chloroform, toluene, and hexane. hi one embodiment, the QDs have an average diameter of 5-7 nm. In another embodiment the QDs have an average diameter of 6.5 nm. These organic-soluble QDs have an quantum yield as high as 60%.

In the third step, preparing aqueous-dispersible CdSe/CdS/ZnS QDs, organic-soluble CdSe/CdS/ZnS QDs (2 mL, 20-40 mg/mL) and mecapto-group containing ligands (MGLs) (2 to 15 mmol) are dissolved in chloroform. In one embodiment examples of suitable MGLs are thiol-substituted carboxylic acids (e.g. mercaptoacetic acid, mercaptosuccinic acid, mercaptopropionic acid, and mercaptoundecanoic acid). The MGLs, associated with (i.e. coordinated to) the quantum dots, are deprotonated by addition of an ammonium hydroxide solution (0.1 to 5 mL) which is added into the mixture with stirring. The mixture is stirred at room temperature overnight. Aqueous-dispersible QDs are separated from the surfactant solution by addition of ethanol and centrifugation. These QDs are easily dispersed in aqueous solutions. Solutions of these QDs can maintain their colloidal stability for more than several months in the dark at 4⁰C. hi general, we have consistently observed that after transferring the QDs into the water phase, a 10 to 30% decrease in QY is observed. Rendering QDs produced using the conventional method aqueous soluble results in a similar decrease in quantum yield. Such decrease did not affect the performance of CdSe/CdS/ZnS QDs produced using the method disclosed herein in bioimaging applications.

QDs produced using the present method have high QY, and good colloidal and optical stability. No aggregation or precipitation was observed even after storing them for more than one year. We have repeatedly used this method to prepare CdSe/CdS/ZnS QDs which are used for cell labeling, cell trafficking, and studying cell interactions, hi comparison, CdSe/ZnS QDs, i.e. CdSe-core nanocrystals with a ZnS shell, prepared using the method described herein (without addition of a cadmium precursor in the second step) are unstable as aqueous dispersions and exhibit significantly lower quantum yields as compared to CdSe/CdS/ZnS QDs prepared using the same method. These CdSe/ZnS QDs were found to be unsuitable for biological applications.

This invention is further described through the following examples, which are to be construed as illustrative and not restrictive in any way.

EXAMPLE 1

Preparation of Organic-Soluble CdSe/CdS/ZnS QDs

The following procedure was found to be optimal in obtaining a high yield of CdSe/CdS/ZnS QDs. Cadmium oxide (6 mmol), oleic acid (6-10 mL) and tetradecylphosphonic acid (2 g) were loaded into a 100-mL three-necked flask. The reaction mixture was slowly heated under an argon atmosphere to 280°C for 20 minutes and TOP-Se (2 mL, 1 M; 0.5 mmol Se in 1.0 mmol trioctylphosphine) rapidly injected into the reaction mixture. Aliquots (7-8 mL) were withdrawn after 1 - 4 minutes. The aliquots were quenched with chloroform. CdSe-core nanocrystals with controllable size can be obtained by withdrawing the aliquots at different times. The nanocrystals were separated from the surfactant solution by addition of ethanol and centrifugation at 15,000 rpm.

Next, a solution containing CdSe-core nanocrystals was prepared by dissolving (0.1- 0.4 g) of CdSe-core nanocrystals in 7 mL of hexane. Separately, cadmium oxide (0.5-2 mmol) and zinc acetate (2-6 mmol) were dissolved in oleic acid (8-10 mL) and trioctylphosphine oxide (TOPO) (5-6 g). The molar ratio of cadmium to zinc generally used for the shell formation are 1:1 to 1:3. The reaction solution was heated to 150°C -190°C for 45 minutes under an argon flow and the CdSe-core-nanocrystal solution injected slowly with stirring. The reaction mixture was held at 150-180⁰C with a needle outlet allowing the hexane to evaporate. After 10 minutes of heating the needle was removed and the reaction mixture heated to 24O⁰C. When the reaction mixture reached 240°C trioctylphosphine (TOP) (1-2 mL)-sulfur (S) (1-2 mmol) solution was added drop wise to the stirred reaction mixture. The reaction mixture temperature was held at 240°C and stirred for 15 to 20 minutes. The thickness of the CdS/ZnS shell is determined by the concentration of TOP-S added into the reaction mixture. Once the desired shell thickness is achieved, the reaction mixture is quenched by addition of a large volume (50 mL) of organic solvent (e.g. chloroform, hexane, or toluene). After quenching the reaction, the organic-soluble QDs were separated from the extra surfactant solution by addition of ethanol and centrifugation at 15,000 rpm. The volume ratio of ethanol to nanocrystal solution 60:40. The CdSe/CdS/ZnS QDs had an average diameter of 6 to 7 nm. The precipitated organic-soluble CdSe/CdS/ZnS QDs were readily re-dispersed in organic solvents such as chloroform, toluene, and hexane. EXAMPLE 2

Preparation of Aqueous-Dispersible CdSe/CdS/ZnS QDs

The following approach was found to be optimal in obtaining high yield of aqueous- dispersible CdSe/CdS/ZnS QDs. Organic-soluble CdSe/CdS/ZnS QDs (~2 mL, 20-40 mg/mL) and mercaptosuccinic acid (3-5 mmol) were dissolved in 10 mL of chloroform. Next, ammonium hydroxide (30% NH₄OH(aq.)) (1-2 mL) was added to the mixture with stirring. The mixture was stirred overnight at room temperature. The aqueous-dispersible QDs were separated from the surfactant solution by addition of ethanol and centrifugation. These CdSe/CdS/ZnS QDs were easily dispersed in water and maintained their colloidal stability for more than several months in the dark at 4°C.

EXAMPLE 3 Properties of CdSe/CdS/ZnS QDs Prepared Using the Present Method

QDs useful for bioimaging can be produced by the present method. The EDS data in Figure 3 shows that cadmium, selenium, sulfur, and zinc are the only elements present in the sample prepared using organic-soluble QDs obtained via the present method. Figure 1 shows a TEM image of organic-soluble QDs with an approximate size of 6.5 nm. Figure 6 shows a TEM of aqueous-soluble QDs. Figure 2 shows data demonstrating the crystallinity of the CdSe-core nanocrystals. Figure 4 shows emission data for organic-soluble QDs produced by three separate runs. The emission maxima for the three runs range from 608- 630 nm.

QDs with these physical and optical qualities are very useful in bioimaging applications. Figure 5 shows an image of a cancer cell labeled with CdSe/CdS/ZnS QDs. The red color in the image is fluorescence from CdSe/CdS/ZnS QDs incorporated in live cells. The oblong shape of the QD-incorporated cells indicates that the cells are alive. If the cells were dead they would have a spherical shape.

Claims

We claim:

1) A method for production of water-dispersible cadmium selenide/cadmium sulfide/zinc sulfide (CdSe/CdS/ZnS) quantum dots (QDs) comprising the steps of: a) combining one or more cadmium compound selected from the group consisting of cadmium chloride, cadmium acetate, cadmium acetylacetonate and cadmium nitrate, with elemental selenium to form CdSe-core nanocrystals; b) combining i) one or more cadmium compound selected from the group consisting of cadmium chloride, cadmium acetate, cadmium acetylacetonate and cadmium nitrate; and ii) one or more zinc compound selected from the group consisting of zinc oxide, zinc chloride, zinc acetylacetonate, zinc nitrate, zinc undecylanate and zinc 2-ethylhexanoate; with a coordinating solvent and a trialkylphosphine oxide to form a precursor solution; c) adding CdSe-core nanocrystals from a) to the precursor solution from b); d) adding elemental sulfur solution to c) to form CdSe/CdS/ZnS QDs; e) combining CdSe/CdS/ZnS QDs from d) with mercapto group-containing ligands to form CdSe/CdS/ZnS QDs with associated mercapto-group containing ligands; and f) subjecting the CdSe/CdS/ZnS QDs with associated mercapto-group containing ligands from e) to a deprotonating reaction to form QDs dispersible in an aqueous solution.

2) The method of claim 1, wherein the cadmium compound is cadmium oxide.

3) The method of claim 1, wherein the zinc compound is zinc acetate. 4) The method of claim 1, wherein the mercapto-group containing ligand is mercaptosuccinic acid.

5) The method of claim 1, wherein the reaction temperatures used in step a) is 190°C to 300°C.

6) The method of claim 5, wherein the reaction temperature used in step a) is 275°C to 285⁰C and the average reaction temperature is 28O⁰C.

7) The method of claim 1, wherein the precursor solution obtained in step b) is heated to 100⁰C to 22O⁰C prior to step c).

8) The method of claim 7, wherein the precursor solution is heated to 150⁰C - 190⁰C.

9) The method of claim 1, wherein the CdSe-core nanocrystals in step c) are added as a solution in hexane, to the precursor solution and the resultant mixture is heated to 150⁰C to 18O⁰C to remove hexane.

10) The method of claim 1, wherein the mixture from c) is heated to 150⁰C to 280⁰C prior to addition of elemental sulfur solution in d).

11) The method of claim 10, wherein the mixture is heated from 235°C to 245⁰C.

12) The method of claim 1, wherein step a) proceeds for a sufficient time to generate CdSe-core nanocrystals with an emission range of 600 ran to 660 nm.

13) The method of claim 13, wherein the CdSe-core nanocrystals have an emission range of 630 nm to 650 nm.

14) The method of claim 1, wherein step d) proceeds for a sufficient time to generate CdSe/CdS/ZnS QDs with an average diameter of 5 nm to 7 nm.

15) The method of claim 14, wherein the CdSe/CdS/ZnS QDs have an average diameter of 6.5 nm.

16) The method of claim 1, wherein the deprotonating reaction in f) comprises addition of an ammonium hydroxide solution.