Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

Summary

In this protocol, the synthesis of Cd-free InP/ZnS quantum dots (QDs) is detailed. InP-based QDs are gaining popularity due to the toxicity of Cd²⁺ ions that may be released through nanoparticle degradation. After synthesis, QDs are solubilized in water using an amphiphilic polymer for use in biomedical applications.

Abstract

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, quantum dots should be highly fluorescent and small/monodisperse in size. While commonly used cadmium-based quantum dots possess these qualities, they are potentially toxic due to the possible release of Cd²⁺ ions through nanoparticle degradation. Indium-based quantum dots, specifically InP/ZnS, have recently been explored as a viable alternative to cadmium-based quantum dots due to their relatively similar fluorescence characteristics and size. The synthesis presented here uses standard hot-injection techniques for effective nanoparticle growth; however, nanoparticle properties such as size, emission wavelength, and emission intensity can drastically change due to small changes in the reaction conditions. Therefore, reaction conditions such temperature, reaction duration, and precursor concentration should be maintained precisely to yield reproducible products. Because quantum dots are not inherently soluble in aqueous solutions, they must also undergo surface modification to impart solubility in water. In this protocol, an amphiphilic polymer is used to interact with both hydrophobic ligands on the quantum dot surface and bulk solvent water molecules. Here, a detailed protocol is provided for the synthesis of highly fluorescent InP/ZnS quantum dots that are suitable for use in biomedical applications.

Introduction

Quantum dots (QDs) are semiconducting nanocrystals that exhibit fluorescent properties when irradiated with light¹. Due to their small size (2-5 nm), which is similar to many larger biomolecules, and ease of biofunctionalization, QDs are an extremely attractive tool for biomedical applications. They have found use in biological labeling, single-molecule live-cell imaging, drug delivery, in vivo imaging, pathogen detection, and cell tracking, among many other uses^2-8.

Cd-based QDs have been most commonly used in biomedical applications because of their intense fluorescence and narrow emission peak widths⁹. However, concerns have been raised due to potential toxicity of Cd²⁺ ions¹⁰ that may be released through degradation of the nanoparticle. Recently, InP-based QDs have been explored as an alternative to Cd-based QDs because they maintain many fluorescence characteristics of Cd-based QDs and may be more biocompatible¹¹. Cd-based QDs have been found to be significantly more toxic than InP-based QDs in in vitro assays at concentrations as low as 10 pM, after only 48 hr¹¹.

The fluorescence emission color of QDs is size-tunable¹. That is, as the size of the QD increases, the fluorescence emission is red-shifted. The size and size dispersity of the QD products can be modified by changing the temperature, reaction duration, or precursor concentration conditions during the reaction¹². While the emission peak of InP QDs is typically broader and less intense than Cd-based QDs, InP QDs can be made in a large variety of colors designed to avoid spectral overlap, and are sufficiently intense for most biomedical applications¹². The synthesis detailed in this protocol yields QDs with a red emission peak centered at 600 nm.

Several steps are taken after synthesis of the QD cores to maintain the optical integrity of the QDs and to make them compatible for biological applications. The surface of the QD core must be protected from oxidation or surface defects that may cause quenching; therefore, a ZnS shell is coated over the core to produce InP/ZnS (core/shell) QDs¹³. This coating has been shown to protect the photoluminescence of the QD product. The presence of zinc ions during InP QD synthesis has been shown to limit surface defects, as well as decrease size distribution¹². Even with the presence of Zn²⁺ in the reaction medium, synthesis of InZnP are highly unlikely¹². After coating, resulting InP/ZnS QDs are coated in hydrophobic ligands such as trioctylphosphine oxide (TOPO) or oleylamine^12,14. An amphiphilic polymer can interact with hydrophobic ligands on the QD surface as well as bulk water molecules to impart water solubility¹⁵. Amphiphilic polymers with carboxylate chemical groups can be used as "chemical handles" to further functionalize the QDs.

This protocol details the synthesis and functionalization of water-soluble InP/ZnS QDs with very intense fluorescence emission and relatively small size-dispersity. These QDs are potentially less toxic than commonly used CdSe/ZnS QDs. Herein, the synthesis of InP/ZnS QDs provides a practical alternative to Cd-based QDs for biomedical applications.

Protocol

1. Synthesis of Indium Phosphide/Zinc Sulfide (InP/ZnS) Quantum Dots

1. Synthesis of Indium Phosphide (InP) Quantum Dot Cores
   1. Fit a 100 ml round bottom, 3-neck, flask with a 12-inch condenser. Add 30 ml oleylamine (OLA), 0.398 g indium (III) chloride (InCl₃), 0.245 g zinc (II) chloride (ZnCl₂) and stir while evacuating at RT using a vacuum for 1 hr. The solution should appear colorless with a white precipitate.
   2. Using a heating mantle with a thermocouple and proportional-integral-derivative (PID) temperature controller, increase the temperature of the solution to 120 °C. Evacuate the solution under vacuum for 20 min to remove low boiling point impurities that may affect core growth.
      Note: While it is possible to use a sand bath and thermometer, using a heating mantle and PID increases the uniformity and reproducibility of the reaction products.
   3. Under inert gas (e.g., N₂), reflux the solution and increase the temperature to 220 °C for 15 min. The InCl₃ and ZnCl₂ completely dissolve, resulting in a pale yellow solution. Allow the temperature to stabilize for 10 min.
   4. Purge a disposable, 3 ml plastic syringe and 4 inch, 22 G needle with nitrogen gas. Using the syringe, quickly deliver 0.5 ml tris(dimethylamino)phosphine (TDMAP) to the InCl₃ solution. The solution temperature decreases slightly and returns to 220 °C. The solution changes from transparent, pale yellow to opaque, black.
   5. After 9.5 min, remove the reaction flask from the heating mantle until the temperature decreases below 200 °C. To protect the integrity of the InP cores, proceed directly to the ZnS coating in step 1.2.1.
2. Synthesis of Zinc Sulfide (ZnS) Quantum Dot Shells
   1. Place the reaction flask from step 1.1.5 on a heating mantle and stabilize the temperature at 200 °C. Slowly add 3.58 g dodecanethiol (DDT) over the course of 15 sec to the solution containing InP QDs. Allow the solution to react for 1 hr.
      Note: ZnS shell thickness can be varied by increasing or decreasing the amount of zinc stearate added in step 1.2.4. Altering the amount of ZnCl₂ or dodecanethiol in steps 1.1.1 and 1.2.1 can significantly impact the quality of QDs by changing the reaction kinetics.
      1. Afterward, remove the reaction flask from the heating mantle and allow the solution to cool to approximately 60 °C.
   2. Once the InP/ZnS solution reaches ~60 °C, add 10 ml hexanes and transfer the entire solution of roughly 45 ml to a 50 ml polypropylene centrifuge tube. Centrifuge the sample (3,000 x g for 10 min) to remove unreacted solid precursors.
   3. Carefully transfer the supernatant to a 250 ml polypropylene centrifuge bottle, add 200 ml acetone, and centrifuge the solution (3,000 x g for 10 min) to precipitate InP/ZnS QDs. This volume can also be split evenly into four 50 ml tubes for centrifugation if a centrifuge with the necessary rotor/accessories is not available. Decant the supernatant and dry the QD pellet thoroughly with nitrogen gas to remove acetone.
   4. Resuspend the QDs in 20 ml OLA using sonication, transfer to a 50 ml round bottom, 3-neck, flask containing 0.474 g zinc stearate, and stir. Evacuate the solution under vacuum for 20 min at RT.
   5. Under nitrogen gas, increase the temperature to 180 °C and allow the reaction to proceed for 3 hr. While there are no noticeable visual changes to the reaction solution that occur during this reaction, adding zinc stearate increases ZnS shell thickness, thus increasing QY by improving surface passivation of QDs^12.. Once the reaction is complete, remove the flask from the heating mantle and allow the solution to cool to approximately 60 °C.
   6. Once the InP/ZnS solution reaches ~60 °C, add 20 ml hexanes and transfer to a 50 ml polypropylene centrifuge tube. Centrifuge the sample (3,000 x g for 10 min) to remove unreacted zinc stearate.
   7. Carefully transfer the supernatant to a 250 ml polypropylene centrifuge bottle, add 200 ml acetone, and centrifuge the solution (3,000 x g for 10 min) to precipitate InP/ZnS QDs. Carefully decant the supernatant and thoroughly dry with nitrogen gas to remove acetone.
   8. Dissolve the InP/ZnS QD pellet in 30 ml hexane. Vortex and sonicate the solution briefly to ensure complete dispersion.
   9. Repeat purification steps 1.2.6-1.2.8 two more times to ensure thorough removal of excess organic ligands. Interactions between the amphiphilic polymer and the QD in step 1.2 can be compromised in the presence of excess ligands.
   10. With calculations detailed by Xie, et al.¹⁶, determine the size and concentration of the synthesized InP/ZnS QDs using UV-Vis Spectroscopy.
       Note: The important spectral characteristic for this analysis is the shoulder of the absorption peak. The wavelength and absorbance value of the maximum of this shoulder is used to calculate the size and the concentration of the QDs, respectively. This reaction typically yields approximately 5 μmol of QDs with an emission peak at 600 nm. To synthesize QDs of different colors, the duration and/or temperature of the reaction can be changed. A longer reaction time and/or higher temperature of the solution results in a red-shifted emission peak. For example, increasing the reaction temperature to 240 °C and maintaining reaction time of 10 min will result in QDs with a 680 nm maximum emissions peak. Likewise, reducing reaction time to 2 min and doubling zinc chloride used will result in QDs with a 470 nm maximum emission peak. These InP/ZnS QDs are stable for at least one month at 4 °C in the dark under inert gas.

2. Water Solubilization of InP/ZnS Quantum Dots Using an Amphiphilic Polymer

1. Water Solubilization
   1. Using the InP/ZnS QDs from step 1.2.10, dilute a portion of the QDs with hexanes to obtain 1 ml of 1 μM QDs.
      1. In a centrifuge tube, transfer 0.25 ml InP/ZnS QDs into each tube. Add 1 ml acetone or methanol to the centrifuge tube and centrifuge (3,000 x g for 10 min). Carefully remove the supernatant and dissolve each precipitate in 1 ml tetrahydrofuran (THF).
      2. Transfer the InP/ZnS QDs dissolved in THF into a 100 ml round bottom flask and dilute with 16 ml THF. To reduce the number of aggregates in solution, sonicate the QDs for 5-10 min.
   2. Dissolve 30 mg poly(maleic andhydride-alt-1-octadecene), 3-(dimethylamino)-1-propylamine (PMAL-d) in 10 ml molecular grade water. Water bath sonication or gentle stirring until the solution is translucent is sufficient to completely dissolve the polymer. The use of vortex or vigorous stirring can produce many bubbles, which hinders interaction of the polymer with the QD. Add the 10 ml polymer solution to the 100 ml round bottom flask containing InP/ZnS QDs in THF.
   3. Evaporate THF from the QD/polymer solution using a rotary evaporator. Place the flask in an ice bath while evaporating to facilitate the interaction between the polymer and QD. Depending on the strength of the vacuum, most THF is evaporated after 10 min and the solution appears turbid.
      1. Once the solution is evaporated to 10 ml, remove the flask from the rotary evaporator and add 30 ml molecular grade water. Return the flask to the rotary evaporator and continue to evaporate to 2 ml. This final evaporation step may take many hours; ensure the ice bath is maintained.
   4. Remove the water-soluble InP/ZnS QDs from the round bottom flask with a pipette. Filter the QD solution using a 3 ml plastic syringe attached to a 0.1 μm nylon syringe filter into a 5 ml centrifuge tube.
   5. Place the QDs into a 20,000 MWCO membrane dialysis unit and dialyze against 0.05 M borate buffer pH 8.5 to remove excess polymer. (Slowly add 0.05 M sodium tetraborate decahydrate to 0.05 M boric acid, with vigorous stirring, until the pH is 8.5 to make this borate buffer solution.) Using a vacuum concentrator, concentrate the QDs in borate buffer to 1 ml.
   6. For storage, purge the solution with nitrogen gas before sealing with Parafilm. The water-soluble InP/ZnS QDs are stable for at least 4 months at 4 °C in the dark.

Results

The uncoated InP cores do not demonstrate substantial visible fluorescence by eye. However, InP/ZnS (core/shell) quantum dots appear to fluoresce brightly by eye under UV irradiation. The fluorescence of InP/ZnS QDs was characterized using fluorescence spectroscopy. The fluorescence spectrum of QDs in hexanes (Figure 1) excited at 533 nm demonstrates one major peak centered at 600 nm with a full width at half maximum (FWHM) of 73 nm. While absorbance (0.2) offset in

Discussion

This protocol details the synthesis of highly fluorescent InP/ZnS QDs that can be used in many biological systems. The QD products synthesized here exhibited a single fluorescence emission peak centered at 600 nm with a FWHM of 73 nm (Figure 1), which is comparable to other previously described syntheses¹². Reaction time and reaction temperature are extremely crucial steps due to their profound effect on QD synthesis quality and repeatability. After solubilization in water, the QDs were determ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Oleylamine	Acros	129540010
Zinc(II) chloride	Sigma	030-003-00-2
Indium(III) chloride	Chem-Impex	24560
Tris(dimethylamino)phosphine	Encompass	50-901-10500
1-dodecanethiol	Acros	117625000
Hexanes	Fisher Sci	H292-4
Acetone	TransChemical	UN 1090
Zinc Stearate	Aldrich Chem	307564-1KG
Tetrahydrofuran	Acros	34845-0010
Molecular Water	Fisher Sci	BP2470-1
Poly(maleic anhyrdride-alt-1-tetradecene), 3-(dimethylamino)-1-propylamine derivative	Sigma	90771-1G
Boric acid	Fisher Sci	BP168-500
Sodium Tetraborate Decahydrate	Fisher Sci	BP175-500
Rhodamine B	Aldrich Chem	R95-3
Nitrogen gas	Airgas	UN1066
Trypan blue	Thermo Sci	SV30084.01
3 ml plastic Luer-lock syringe	BD	309657
Luer-lock Needle	Air-Tite	8300014471	4 inch, 22 gauge
50 ml polypropyene centrifuge tube	Falcon	352098
250 ml centrifuge bottle	Thermo Sci	05-562-23	Nalgene PPCO
5 ml centrifuge tubes	Argos-Tech	T2076
1.5 ml microcentrifuge tubes	Bio Plas	4150
0.1 μm Syringe filter	Whatman	6786-1301	Puradisc 13 mm nylon filter
Slide-A-Lyzer MINI Dialysis Unit	Thermo Sci	69590	20,000 MWCO
Rotary Evaporator	Heidolph
Centrifuge 5072	Eppendorf		Swinging Bucket with 50 ml tube adapters
Lambda 650 UV/VIS Spectrometer	Perkin Elmer		UV-Vis Spectrophotometer
LS 55 Fluorescence Spectrometer	Perkin Elmer		Fluorometer
Axio Observer.A1	Zeiss		epifluorescence microscope
AxioCam MRm	Zeiss		CCD Camera
Tecnai TF20 Microscope	FEI		Transmisison Electron Miscroscope
TEM Eagle CCD	FEI		TEM CCD Camera
NanoBrook Omni DLS	Brookhaven		Dynamic Light Scattering Instrument