On the Characterization of the Surface Chemistry of Quantum Dots

(12, 13) Although very efficient, NMR characterization of the colloidal QD–ligand ... (15) using thermogravimetry (2.5 stearate/nm2). .... nl402192d...
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On the Characterization of the Surface Chemistry of Quantum Dots Fabien Knittel,† Edmond Gravel,† Elsa Cassette,‡ Thomas Pons,‡ Florence Pillon,† Benoit Dubertret,*,‡ and Eric Doris*,† †

Service de Chimie Bioorganique et de Marquage, CEA, iBiTecS, 91191 Gif-sur-Yvette, France Laboratoire de Physique et d’Etude des Matériaux, UMR8213 du CNRS, ESPCI, 10 rue Vauquelin, 75005 Paris, France

‡

S Supporting Information *

ABSTRACT: The interaction of ligands with the surface of quantum dots (QD) was studied using tritiated oleic acid as an ultrasensitive reporter. The use of labeled oleic acid not only permitted to quantify the number of ligands attached to the surface of QDs of various sizes but also enabled the investigation of the relative affinity of different ligand types for the nanocrystal’s surface. KEYWORDS: Ligand exchange, quantum dots, surface chemistry, tritium

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conceived that isotopic labeling could provide an interesting alternative to existing methods as radioactive tracers can easily be detected in minute amounts. Our strategy hinges on the use of tritium as a highly sensitive reporter for the quantification of ligands bonded to the QDs. Radioactive labeling with tritium (3H) offers many advantages, for example, (i) the 3H nuclei is a highly sensitive reporter and trace amounts as little as 10−10 Ci of tritium can easily be detected by standard scintillation counting techniques (this value corresponds to less than picomoles of the ligand to be detected, depending on the specific acitivity of the latter), and (ii) introduction of the 3H label in the compound of interest can be done without altering its chemical structure. In this communication, we investigate the quantification of OA ligands on QDs of various sizes and evaluate their displacement by competing ligands. Six different batches of zinc blende CdSe QDs were synthesized in octadecene at 240 °C from cadmium myristate and selenium powder.14 The as-produced QDs were capped with oleic acid ligands and were characterized using optical absorption spectroscopy and transmission electron microscopy (see Supporting Information). The above syntheses afforded nanoparticles of tetrahedral shape with heights comprised between 3.4 and 6.9 nm (see Supporting Information Figure S1). This approach provided a simple model system in which QDs are only stabilized by carboxylate type (oleate and myristate) ligands that are complexed to cadmium surface atoms through electrostatic interactions. In parallel to the synthesis of the QDs, 3H-labeled oleic acid 2 was synthesized by partial reduction of stearolic acid (1) under tritium gas atmosphere in the presence of the palladiumbased Lindlar catalyst (Scheme 1a). The use of a lead-poisoned catalyst was required to avoid over-reduction of the double

uantum dots (QDs) are crystalline nanoparticles that often possess unique optical properties including tunable emission, high quantum yields, and improved photostability compared to organic fluorophores.1,2 QDs are classically made of an inorganic semiconductor particle coated with organic surface ligands and have found numerous applications in domains ranging from electronics to bioimaging.3−10 The coating ligands are usually incorporated in the course of the QD synthesis and are important components as they influence the growth and size of the nanoparticles, provide colloidal stability and passivate surface traps. Among the various ligands that have been used in QD synthesis, phosphines, amines, and carboxylic acids are the most widespread species. A key aspect of the surface-bound ligands is their dynamic character as they are interchangeable, hence allowing the replacement of pendant groups and providing the interface to various applications. The primary benefit of ligand exchange is to make nanoparticles suitable for different media (e.g., water) or applications (e.g., in vivo imaging). While most studies usually focus on the improvement of the properties of the core of QDs, there is also an increasing interest in understanding their surface chemistry as the peripheral organic layer can account for specific physicochemical properties. Experimental techniques that have been used so far to investigate the nanoparticle-bound ligands include thermogravimetric analyses, X-ray photoelectron spectroscopy, infrared spectroscopy, photoluminescence spectroscopy, and nuclear magnetic resonance (NMR).11 For example, Hens et al. recently used the latter technique to study the nature of the interaction of oleic acid (OA) with QDs and the number of OA-ligands bonded to their surface.12,13 Although very efficient, NMR characterization of the colloidal QD−ligand complexes necessitates relatively high concentrations of material to achieve adequate signal as proton resonances of surface-bound species are often broadened (because of chemical-shift heterogeneity and/or decreased T2 relaxation time). With this problem in mind, we © 2013 American Chemical Society

Received: June 17, 2013 Revised: October 3, 2013 Published: October 10, 2013 5075

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Scheme 1. (a) Synthesis of 3H-labeled OA Ligands and (b) Protocol Used for Their Quantification at the Surface of QDs

Figure 1. Evolution of the number of ligands per QD as a function of the square of the QD height (h).

observed that the number of incorporated OA ligands was proportional to the square of the QD height. This result strongly suggests that QDs of different sizes retain similar shapes and comparable surface ligand densities. When assuming a constant tetrahedral shape with {110} and {100} faces as observed with larger size samples, we estimated an average ligand density of ca. 0.8 OA/nm2. This value is lower than those measured by Hens et al.13 using NMR spectrometry (4.6 OA/ nm2) and Brutchey et al.15 using thermogravimetry (2.5 stearate/nm2). The observed differences may originate from different synthetic schemes of the starting QDs and therefore different surface compositions. More specifically, Hens’ QDs exhibited higher Cd/Se ratio (1.23 → 23% excess Cd) than the QDs used in our study (1.05−1.10 → 5−10% excess Cd). Cadmium in excess is located at the surface of the QDs and involved in the anchoring of oleate ligands. Since our QD samples are less enriched in surface Cd atoms, one could expect a lower fraction of stabilizing OA ligands. As recent reports demonstrated that methanol could displace oleate surface ligands by proton exchange,16 we investigated whether the two-round methanol precipitation used in our procedure significantly altered the number of oleic acid bonded to the QDs. Accordingly, after the typical ligand exchange procedure, 3H-OA in excess were removed by dialysis against hexane (instead of double MeOH precipitation). This experiment showed no difference in ligand density in the recovered QD sample, suggesting that the initial mild precipitation in MeOH did not significantly remove oleate ligands from the QD surface. However, a third precipitation cycle in methanol did lead to a dramatic ligand density decrease. In addition to the quantification of ligands, quantum dots coated with tritium-labeled OA were also used as probes to evaluate the strength of competing ligands in regard to their ability to displace OA from the QD surface. As ligand exchange (i.e., displacement of the initial coating of 3H-OA by competing nontritiated species) should lead to an overall decrease of the radioactivity borne by the QD, a relative scale of “strength” could be established for various exogenous ligands. QDs capped with tritium-labeled OA were thus incubated with nonlabeled ligands incorporating different complexing groups such as carboxylic acid, amine, phosphine, or phosphine oxide (Figure 2a) at variable concentrations. After the exchange procedure, the QD solutions were purified as described above and the fraction of tritiated ligands remaining on the surface of the QD was assessed by scintillation counting (Figure 2b).

bond that would have led to fully reduced stearic acid. The crude mixture was worked-up by filtering-off the catalyst, evaporating the solvent, and purifying the product by HPLC. 3 H-labeled oleic acid was recovered pure with a specific activity of 42.4 Ci mmol−1 as determined by mass spectrometry (maximum theoretical incorporation = 58 Ci mmol−1). This specific activity corresponds to an average of ca. 1.5 tritium atoms incorporated per molecule of oleic acid. To minimize autoradiolysis, the labeled oleic acid was dissolved in toluene at a concentration of 1 mCi mL−1 and stored at −20 °C. The stock solution of labeled oleic acid was then diluted with nonlabeled oleic acid to reach a specific activity of 3.5 × 10−3 Ci mmol−1 and used as such in the ensuing ligand exchange experiments. In order to evaluate the number of OA ligands that are bonded to the QDs, various ligand exchange experiments were performed using an excess of the isotopically labeled OA (Scheme 1b). Different conditions were tested to optimize the ligand exchange procedure. The amount of labeled OA per QD surface unit was progressively increased and ligand exchange saturated when 3500 OA per nm2 of QDs were present in solution (Supporting Information Figure S5) as no further incorporation of ligands was observed when working at higher concentrations of labeled oleic acid. QDs were then recovered by two rounds of precipitation in methanol and redispersed in toluene. The QD concentration and the number of ligands were then assessed using UV−vis absorption and liquid scintillation counting, respectively. The absorption properties of the QDs showed no significant shift throughout the ligand exchange procedure, indicating no alteration of their inorganic core. Having optimized the amount of ligands to be used in excess and the incubation time with the QDs (i.e., 240 min), we next investigated various sizes of nanocrystals in regard to the number of ligands bonded to their surface (Figure 1). We 5076

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difference in reactivity can be rationalized by the formation of relatively strong TOP-Se bonds at the QD surface, facilitating the stripping of cadmium oleate complexes. It may also result from the acidic phosphorus-containing impurities that are present in TOP samples.19 Taken together, these exchange experiments allowed us to establish a relative scale of OA displacement strength for the different ligands, that is, stearic acid ∼ oleic acid > trioctylphosphine > octadecylamine > oleylamine > trioctylphosphine oxide, which is the weakest of the investigated ligands (Figure 2c). In conclusion, a novel and versatile method for the quantification of specific ligands at the surface of nanoparticles is reported. We demonstrated that the surface density of oleic acid ligands on our CdSe nanocrystals remains constant during the nanocrystal growth. We also evaluated the efficiency of different ligand exchange reactions and showed that both Xand L-type ligands were able to efficiently displace oleates from the QD surface, either as isolated ligands or as metal complexes. Ligand quantification using 3H-labeling thus appears as a very sensitive technique to characterize interactions with inorganic nanoparticles. The incorporation of tritiated ligands in the course of the synthesis of the nanocrystals using, for example, labeled Cd-oleate precursors, is currently under investigation and should allow further quantification of the exchangeable ligand fraction.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed experimental protocols and additional data. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Figure 2. (a) Chemical structures of the different competing ligands; (b) amount of labeled OA remaining at the QD surface after exchange with an excess of 53 (purple), 106 (green), 266 (red), or 2128 (blue) competing ligands per nm2 of QD surface; (c) relative OA displacement strengths of the different ligands, taking OA as reference.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: (B.D.) [email protected]. *E-mail: (E.D.) [email protected]. Notes

The first observation is related to the steady decrease of the radioactivity borne by the QD upon increasing the amount of competing ligands from 53 to 2128 ligands per nm2. This tendency was observed for all the tested ligands. However, trioctylphosphine (TOP) exhibited a much weaker OA displacement ability at low concentrations (i.e., 106 and 53 ligands per nm2). This loss of affinity for the QD surface can likely be rationalized by the oxidation of TOP into less affine trioctylphophine oxide (TOPO) ligands in the course of the experiment. Oleic acid and stearic acid (SA) provided nearly similar exchange efficiencies, indicating that the double bond played little to no role in the exchange. The second observation is that L-type ligands such as primary amines (oleylamine (OAm) or octadecylamine (ODAm)) as well as trioctylphosphine (TOP) and its corresponding oxide (TOPO)) were also able to displace the original OA ligands. Hens et al. have reported that for the ligand exchange reaction to take place, the incoming ligand molecule has to transfer an acidic proton to the bonded oleate unit.13 However, in the case of the abovementioned L-type ligands there are no acidic protons. Although no significant shift was detected in the absorption spectrum after exchange with L-type ligands (see Supporting Information Figure S8), one can assume that the original ligands may be displaced in the form of cadmium oleate complexes.17,18 We also observed that trioctylphosphine was much more efficient than trioctylphosphine oxide in displacing OA ligands. This

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The “Service de Chimie Bioorganique et de Marquage” belongs to the Laboratory of Excellence in Research on Medication and Innovative Therapeutics (LabEx LERMIT). The “Agence Nationale de la Recherche” (ANR) is acknowledged for financial support.

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REFERENCES

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