Enhancing the physicochemical and photophysical properties of small (<2.0 nm) CdSe nanoclusters for intracellular imaging applications

Abstract

The chemical properties of surface passivating ligands that surround nanoclusters (NCs) are extremely important for controlling their structural, physicochemical, and photophysical properties. However, to investigate these properties in various environments, it is important to be able to dissolve the ligand-coated NCs in a wide range of polar and nonpolar solvents. We have developed a direct synthesis and purification procedure for poly(ethylene glycol) thiolate-protected ultra-small (<2.0 nm diameter) CdSe NCs, which have such solubility characteristics. These CdSe NCs were characterized by UV-vis absorption, photoluminescence, and X-ray photoelectron spectroscopy (XPS), as well as mass spectrometry. Analyses by XPS confirmed the formation of surface Cd-rich NCs and mass spectrometry confirmed a stoichiometric core with mass of 6.5 kDa. The NCs were synthesized in aqueous medium and purified using a simple solvent extraction method, and the organic phase-extracted CdSe NCs were readily soluble in a wide range of polar and nonpolar organic solvents including acetonitrile, ethanol, chlorobenzene, dichloromethane, and chloroform, as well as in water. The diverse solubility properties of poly(ethylene glycol) thiolate-protected ultra-small CdSe NCs allowed us to perform a post synthetic surface ligand treatment with triphenylphosphine in an organic solvent. The post synthetic treatment enhanced the photophysical properties of these ultrasmall NCs and resulted in an ∼8 fold increase in fluorescence quantum yield. These bright yellow, mixed ligand-coated CdSe NCs were then used for intracellular imaging studies for the first time due to their unique physicochemical properties. We also found that the penetration ability of mixed ligand-coated CdSe NCs through fibroblast cell membranes and the fluorescence properties of the NCs inside the cell were both strongly poly(ethylene glycol) chain length dependent.

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Introduction

Synthesis and isolation of quantum dots (QDs) of different size, shape, and composition have been of tremendous interest in modern nanotechnology-based research.^1–9 Due to their unique photophysical and physicochemical properties, these QDs have successfully been used in the fabrication of efficient photovoltaic devices,^5,10 light-emitting diodes,^11–13 and photodetectors.^14,15 In this context, ultra-small nanoclusters (NCs) are a special type of semiconductor nanomaterial because they display sharp absorption peaks as well as deep-trap^16–18 or a combination of deep-trap and band-edge photoluminescence (PL).^19–23 Furthermore, these ultra-small NCs bridge the gap between molecules and QDs by having discrete energy levels with molecule-like highest occupied (HOMO) and lowest unoccupied (LUMO) molecular orbitals. Additionally, at the ultra-small size regime the majority of the atoms reside on the surface of the NCs; therefore, fundamentally new photophysical properties will originate at the NC core–surface ligand interface that are not possible for traditionally studied QDs. Several synthetic methods have been developed to prepare ultra-small II–VI^{16,17,19–29} and IV–VI³⁰ NCs and further tune their PL properties by controlling their surface ligand chemistry.³¹ The current methods of preparing ultra-small NCs involve long-chain hydrocarbon-containing aliphatic amines, phosphine, phosphine oxide, phosphonic acids, or mixture thereof as stabilizing ligands.^{5,10,16–18,20,22,23,25,32–40} These ligand-passivated NCs have shown promise in the fabrication of solid-state light-emitting devices,^21,41 but to our knowledge ultra-small NCs have yet not been used for intracellular imaging applications.

QDs and ultra-small NCs passivated by long hydrocarbon chain-containing ligands are only soluble in organic solvents, which are incompatible with biological systems. Encapsulation of hydrophobic ligand-coated NCs with phospholipids or PEGylated polymers has been used to prepare water soluble NCs.^32,42–45 Additionally, post-synthetic ligand exchange reactions on these QDs with various thiolate ligands have been widely used to enhance the solubility of ligand-coated QDs in aqueous medium for biological imaging.^7,45,46 Thiol forms a strong metal–thiolate bond, which increases the stability of QDs in various solvents. However, thiols are also known to quench the PL quantum yield (PL-QY) of either QDs or ultra-small NCs due to trapping of photogenerated holes that prevents radiative recombination of excitons.⁴⁷ This result is in agreement with previous aqueous phase synthesis and characterization of ultra-small CdSe NCs^24,26,48 in which PL-QYs have not been reported, most likely due to extremely low values. Additionally, thiols only bind with surface metals (e.g., Cd) leaving the surface Se sites free, which creates trap states resulting in faster electron–hole recombination and low PL-QY.

To fully exploit their properties, a systematic manipulation of the surface ligand chemistry of ultra-small NCs is required, which should provide the following advantages: (1) long-term stability in both aqueous and organic solvents, (2) solubility in organic solvent that facilitates an organically soluble ligand treatment to minimize trap states and an increase in PL-QY without removing the original ligands, (3) solubility in aqueous medium for intracellular imaging studies, (4) reduction of nonspecific interaction in biological systems, and (5) penetration of NCs through cell membranes facilitated by small hydrodynamic radii. Taken together, the enrichment of above-mentioned photophysical and physicochemical properties of ligand passivated ultra-small NCs will facilitate their potential application in the field of nanobiotechnology.

This article describes a new method of preparation and isolation of ultra-small (<2.0 nm in diameter) poly(ethylene glycol) (PEG)-thiolate-protected CdSe NCs with substantially enhanced solubility and fluorescence properties, which display all the above mentioned characteristics. The NCs displayed a sharp absorption peak that resembled the so-called “magic-sized” CdSe NCs.^{16,26,49–51} Furthermore, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis confirmed that our NCs contained 6.5 kDa core mass. These organic phase-extracted and purified CdSe NCs were readily soluble in a wide range of polar and nonpolar organic solvents as well as in water. Such unique solubility of the PEG-thiolate-protected CdSe NCs allowed us to perform post synthetic ligand treatment and modification with triphenylphosphine (TPP) to further enhance their PL-QY up to 8-fold, which resulted in a bright yellow PL. From X-ray photoelectron spectroscopy (XPS) we confirmed that the CdSe NC surface was coated with mixed thiolate and TPP ligands. The presence of PEGs on the NC surface and the bright yellow PL facilitated the utilization of such ultra-small NCs as inorganic fluorophores for in vitro cell imaging for the first time. Importantly, we have found that the PEG chain length (PEG_n, n = number of glycol units, 6 and 18) significantly modulated the penetration of mixed PEG_n-thiolate- and TPP-protected CdSe NCs through the cell membranes, as well as their PL properties inside fibroblast cells. To the best of our knowledge, this is the first example where ultra-small CdSe NCs were utilized for intracellular imaging studies.

Experimental section

Materials

CdSO₄·8/3H₂O (>99%), selenium metal (99.99%), triphenylphosphine (99%), different chain length poly(ethylene glycol) methyl ethers (PEG_n, n = glycol unit = 6 and 18), p-toluene sulfonyl chloride (>99%), thiourea (>99%), anhydrous acetonitrile (CH₃CN, >99.8%), hexanes (99%), ethanol (98.5%), chloroform (>99%), and dichloromethane (DCM, >99%) were purchased from Aldrich and used without further purification. Organic solvents were purged with N₂ for 30 min prior to use. A 0.25 M aqueous solution of Na₂SeSO₃ was prepared according to the literature.²⁶ PEG_n-thiols were synthesized following a published procedure (see ESI†).⁵²

Optical spectroscopy, electron microscopy, and mass spectrometry measurements

UV-vis absorption spectra were collected using a Varian Cary 50 UV-vis spectrophotometer over a range of 800–300 nm. Prior to the sample measurements, the baseline was corrected with pure solvent. The emission spectra were acquired using a Cary Eclipse fluorescence spectrophotometer from Varian Instruments. ¹H NMR was recorded on a Bruker AVANCE III 500 instrument at 500 MHz. Typically ∼2 mg of sample were dissolved in 0.6 mL of CD₂Cl₂ at room temperature and a minimum of 1000 scans were collected. TEM analysis was performed using a JEOL 3200FS-JEM instrument at 300 kV beam energy. The TEM sample was prepared inside a glovebox by placing a drop of CdSe NCs in CH₃CN onto a lacey carbon-coated copper grid (Electron Microscopy Science), and excess solution was removed by wicking with a Kimwipe to avoid particle aggregation.

The PL-QYs of the synthesized PEG-thiolate-protected CdSe NCs before and after TPP treatment were calculated via a comparison technique using coumarin-30 as a standard fluorophore. Coumarin-30 exhibits UV-vis absorption maximum at 407 nm and an emission maximum at 482 nm when excited at 380 nm with a QY of 55.3% in acetonitrile.⁵³ All samples were prepared in DCM and the optical density of the samples was kept to a similar level (∼0.08–0.1). The emission data were collected from 350–750 nm and the area of the PL peak was determined between 400–700 nm. The following equation was used to calculate the QY of the CdSe NCs:¹⁷

here QY_NC, A_NC, and E_NC represents the calculated quantum yield, measured absorbance, and integrated emission intensity of the CdSe NCs. QY_STD is the quantum yield of coumarin-30 and the η_s refer to the refractive indices of the two solvents.

Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) measurements were performed using a Bruker Autoflex equipped with a nitrogen laser. 2-[(2E)-3-(4-tert-Butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB) was used as the matrix. CdSe nanoclusters were dissolved in DCM and mixed with DCTB in THF (20 nM). Five μL of the freshly prepared matrix solution was mixed with 1 μL of sample, vortexed, and then 2 μL of the mixture was applied to the target and air dried.

Synthesis of PEG₆-thiolate-protected CdSe NCs

Synthesis was performed using nanopure water under an N₂ atmosphere. In a typical synthesis, stock solutions of CdSO₄·8/3H₂O (1 M) and Na₂SeSO₃ (0.25 M) were prepared in water. In a 100 mL two-neck round-bottom flask, 30 mL of N₂-purged water was mixed with 120 μL CdSO₄·8/3H₂O and the required amount of PEG₆-SH. The reaction mixture was stirred at room temperature under N₂ for 30 min. Next, 1.0 M NaOH was added dropwise to the reaction mixture under gentle stirring until a stable pH of 11.5 was achieved. The solution was heated to 30 °C for 5 min with stirring, then 0.36 mL of Na₂SeSO₃ was quickly injected, and the formation of CdSe nanoclusters was followed by UV-vis spectroscopy. The final concentrations of the reactants were calculated to be 4 mM CdSO₄, 32 mM PEG₆-thiol, and 3 mM Na₂SeSO₃. Under identical reaction condition and mole ratios of reagents, <2.0 nm diameter CdSe NCs were synthesized using PEG₁₈-thiolate as the surface passivating ligand.

Purification of PEG₆-thiolate-protected CdSe NCs

The PEG₆-thiolate-protected CdSe nanoclusters were purified by solvent extraction and precipitation. The prepared aqueous nanocluster solution was transferred to a 500 mL separatory funnel. A mixture of DCM and isopropanol (ratio 1.0 : 0.5) was added to the nanocluster solution and was shaken vigorously. The solution was then allowed to stand in the dark until two distinct layers appeared, where the top layer was completely clear and the bottom layer was yellow. The yellow solution was collected and then brought to dryness using a rotary evaporator. The resulting yellow material was then redissolved in a minimum volume of DCM and precipitated by adding hexane. The hexane precipitation was performed at least three times. The solid was then collected by centrifugation, dried under high vacuum, and stored under N₂ in the dark. The dried samples were redissolved in various N₂-purged solvents prior to study. The similar strategy was adopted to purify PEG₁₈-thiolate-passivated CdSe NCs.

Post-synthetic surface modification of PEG_n-thiolate-protected CdSe NCs (n = 6 and 18)

Using the extinction coefficient 27 560 M⁻¹ cm⁻¹ as determined from literature method,⁵⁴ a 0.215 mM stock solution of PEG_n-thiolate-protected CdSe NCs was prepared in DCM. To the solution, 0.56 g (2.15 mmol) TPP was added. The reaction mixture was stirred under N₂ atmosphere until a stable PL intensity was observed and then purified using the hexane precipitation method described above. For the time-dependent PLQY measurement, small quantities of the reaction mixture were removed through syringe at different time intervals. After reaching the stable PL-QY, the CdSe NCs protected with both TPP and PEG_n-thiolate were purified by hexane precipitation as described above. No significant difference in the QY before and after hexane precipitation was observed.

Live cell imaging studies

Mouse 3T3 fibroblast cells transfected with green fluorescence protein–actin vectors (Clontech Laboratories) were cultured in low glucose DMEM with 10% (v/v) bovine serum with 1% penicillin–streptomycin antibiotic at 37 °C under 5% CO₂ atmosphere. Purified TPP-treated, PEG_n-thiolate-protected CdSe NCs were diluted in culture medium to 100 nM and carefully micropipetted into the solution containing the fibroblasts, which had been placed on coverslips one day before imaging. Cell images were acquired by an Olympus FV1000 confocal microscope 60× objective equipped with a mercury lamp source (USHIO Inc. Japan), a 405 nm excitation laser, a 546 nm wavelength filter, and an incubation system (Tokai Hit, Tokyo, Japan) that controlled the temperature and CO₂ as mentioned above. Image acquisition, processing, and analysis were done with FV-ASV software (Olympus American, PA). For cytotoxicity studies TPP treated PEG₁₈-thiolate-protected CdSe NCs were aided into fibroblast cells and incubated for 24 h. The cell viability was then semiquantitatively measured using a two-dye LIVE/DEAD assay. Approximately 200 cells were counted and the percentage viability was determined over 500 nM, 100 nM, 1 μM, 50 μM, and 100 μM concentrations.

Results and discussion

Synthesis and photophysical properties of PEG₆-thiolate-protected CdSe NCs

The formation of PEG₆-thiolate-passivated ultra-small CdSe NCs was monitored through UV-vis absorption spectroscopy over a period of 7 h as illustrated in Fig. 1. Immediately after the addition of freshly prepared Na₂SeSO₃ (1 min), the spectrum showed two broad features, which were a shoulder at ∼350 nm and a peak near 390 nm. The intensity of the lowest energy (first absorption) peak continued to grow quickly with concurrent shifting towards higher wavelength. After 30 min, a well-defined absorption peak near 425 nm had developed. Over the course of the next 6.5 h, the 425 nm peak continued to grow and red-shift to 429 nm while improving in sharpness. Over the same 7 h the peak at 350 nm also grew. However, the increase in its intensity was comparably slower than that of the peak at 429 nm. The growth of the NCs was completed over 7 h, and after this no detectable changes in peak intensity and position were observed over a 24 h period.

Fig. 1 (A) Time-dependent absorption spectra following formation of PEG₆-thiolate-protected CdSe NCs. The maximum intensity of the lowest energy absorption peak was observed ∼7 h after Na₂SeSO₃ addition. (B) The emission spectra of the crude reaction mixture after 7 h of reaction. The excitation wavelength was 365 nm.

The aqueous solution of PEG₆-thiolate-protected CdSe NCs, which displayed a stable absorption peak at 429 nm after 7 h, was analyzed by PL spectroscopy (Fig. 1B). The spectrum displayed a broad emission band centered at 550 nm attributable to surface trapped electrons and holes.^16,21 The broad emission property of these PEG₆-thiolate-passivated CdSe NCs is in agreement with previous literature, where stabilizing ligands such as TOP,⁵⁵ TOPO,¹⁷ or a mixture of aliphatic amines and phenyl carboxylate¹⁶ were used as surface passivating ligands.

In order to compare the optical properties with the size of the SCNCs obtained at the end of the synthesis, the crude CdSe NC solution was deposited on a TEM grid and analyzed. ESI-Fig. 1† illustrates a representative TEM image of the NCs which appear to be aggregated on the TEM grid. This aggregation could be due to the viscous nature of the PEGs and/or the drying process on the TEM grid. The diameter of the NCs determined from TEM analysis was 2.0 ± 0.7 nm, which is slightly higher than the size calculated by an empirical formula (1.7 nm) based on the lowest energy absorption peak of the CdSe NCs.⁵⁴ The difference in size could be due to skewing from the aggregation of NCs as mentioned above.

Purification and analytical characterization of PEG₆-thiolate-protected CdSe NCs

PEGs are soluble in a wide range of polar and nonpolar organic solvents. Because of their diverse solubility properties it is extremely difficult to remove unbound ligands (e.g., PEG-thiol in solution) from the ligand-protected NCs (e.g., PEG-thiolate-protected NCs). However, it is critical to obtain pure NCs for quantitative characterization of photophysical properties and future applications. Previously, it was shown that ω-functionalized alkylthiolate-protected CdSe QDs can be purified by addition of isopropanol into an aqueous solution of QDs.⁴⁸ Following the same protocol we were unable to precipitate our PEG₆-thiolate-protected CdSe NCs from the aqueous solution. This is likely because PEG₆-thiolate-protected CdSe NCs are also significantly soluble in isopropanol. Thus we deduced and demonstrated a unique approach to purify the NCs, namely a solvent-induced phase transfer followed by an additional precipitation step.

The experimental section outlines in detail the purification procedure of PEG₆-thiolate-protected CdSe NCs. It is also important to mention that once the NCs are transferred into organic solvent, further purification steps via solvent-induced precipitation can be performed without causing aggregation of the NCs. Additionally, the aqueous solution of the solvent-extracted NCs had a pH of 7.4, whereas the crude reaction mixture had a pH of 11.5. This experimental result suggested that the purified NCs were free from excess salts and stabilizing ligands. This result is significant in the context of previously reported solvent-induced precipitation techniques where both NCs and inorganic salt precipitated simultaneously. Our purified PEG₆-thiolate-protected CdSe NCs are soluble in a wide range of organic solvent as well as in water. Fig. 2A shows a photograph of dissolved PEG₆-thiolate protected CdSe NCs in different solvents taken under normal laboratory light.

Fig. 2 (A) Photograph of PEG₆-thiolate-protected CdSe NCs dissolved in different solvents. (B) Absorption spectra of PEG₆-thiolate-protected CdSe NCs in different solvents. The lowest energy absorption peak was at 430 nm. (C) TEM image of purified PEG₆-thiolate-protected CdSe NCs. The NCs are ∼1.6 nm in diameter. Scale bar is 10 nm.

The absorption spectra of purified PEG₆-thiolate-protected CdSe NCs in few selected solvents are shown in Fig. 2B. Importantly, no noticeable differences in either peak position or peak shape were observed. For example, the crude PEG₆-thiolate-protected CdSe NCs (before solvent extraction and purification) displayed a first absorption peak at 429 nm in water and after purification the position of the peak was 430 nm. The purified PEG₆-thiolate-protected CdSe NCs were dissolved in CH₃CN, drop-casted on a TEM grid, and analyzed. Fig. 2C illustrates a representative image of the well-dispersed nanoclusters with size of 1.6 ± 0.2 nm and is in good agreement with the size calculated by the empirical formula of 1.7 nm.⁷⁰

As we mentioned before, it is extremely important to obtain pure NCs in order to properly assess their photophysical properties. Purified PEG₆-thiolate-protected CdSe NCs were analyzed by ¹H NMR, and the spectrum showed broad peaks in terminal methyl (–CH₃: 3.30 ppm), glycol (–O–CH₂–CH₂: 3.71–3.46 ppm), and methylene protons attached to the sulfur (–S–CH₂: 2.65 ppm) of the PEG₆-thiolate (see Fig. 3). The broad peaks can be attributed to a combination of spin-spin relaxation broadening, dipole broadening, and a distribution of chemical shifts,^56,57 which is commonly observed for long-chain aliphatic ligands used for surface passivation of NCs.^16,58 Additionally, the purified CdSe NCs were also analyzed by powder X-ray diffraction (XRD) (ESI-Fig. 2†). The diffraction features at 25.4° and 45.7° appeared broad, which indicated the presence of ultra-small CdSe NCs.^16,48 The NC size was 1.7 nm by calculation from the full width at half maximum of the 25.4° peak by the Scherrer formula. Thus the calculated diameter from XRD data is in agreement with the high-resolution TEM analysis of 1.6 nm.

Fig. 3 Comparison of ¹H NMR of pure PEG₆-thiol (lower panel) and purified PEG₆-thiolate-protected CdSe NCs (upper panel). The spectra were recorded in CD₂Cl₂.

The PEG-thiolate-protected CdSe NCs were analyzed by MALDI-TOF MS using a DCTB matrix to determine their core composition. DCTB matrix is routinely used for MS analysis of thiolate-protected gold nanoparticles to determine their composition.^59,60 The mass spectra in Fig. 4 show detailed information about the core composition of purified PEG₆-thiolate-protected CdSe NCs. The spectra obtained at low laser power exhibited a sharp peak centered at m/z 6505.1 which corresponds to the (CdSe)₃₄ crystalline core (calculated m/z = 6506.6). The slight broadness of the peak likely indicates the presence of a contribution from a (CdSe)₃₃ core along with the (CdSe)₃₄ core.

Fig. 4 MALDI-TOF MS spectra of PEG₆-thiolate-protected CdSe NCs at two different laser powers. CdSe NCs were dissolved in DCM and mixed with DCTB matrix in THF with ∼1 : 1000 NCs : matrix ratio.

At higher laser power the peak at m/z 6505.1 became more intense along with the appearance of few other low-mass peaks. The position of these peaks corresponds to (CdSe)₂₇, (CdSe)₂₃, (CdSe)₁₉, and (CdSe)₁₃ cores. This suggests that the (CdSe)₃₃ and (CdSe)₃₄ cores underwent fragmentation at high laser power.⁴⁹ The presence of (CdSe)₁₉ and (CdSe)₁₃ cores also suggested that CdSe nanoclusters with specific core compositions of (CdSe)₁₃, (CdSe)₁₉, (CdSe)₃₃, and (CdSe)₃₄ possess high stability.⁵¹ Beside fragmentation to (CdSe)₁₉ and (CdSe)₁₃ cores at high laser power and increased peak sharpness at m/z 6505.1, no low intensity higher-mass peaks were observed. Therefore, it is clearly evident from MALDI-TOF MS analysis that our samples contained predominantly (CdSe)_33/34 cores. Since thiolate is a X-type ligand and only binds with Cd-sites,⁶¹ therefore one would expect that this ligand capped nanolcuster should have stoichiometry core with Cd-rich surface to maintain the overall charge balance. The XPS analysis indeed confirmed that the Cd : Se ratio is 1.35 : 1.0. However, in the MALDI-TOF-MS analysis we were unable to determine the total mass of the ligand-coated CdSe NC. This could be due to fragmentation of the Cd-bound thiolate-ligand leaving a core mass of ∼6.5 kDa.

Using identical molar ratio of reagents, we also synthesized the longer PEG chain length PEG₁₈-thiolate-protected CdSe NCs to investigate the ligand effects on the physicochemical and photophysical properties of ultra-small NCs. ESI-Fig. 3† illustrated the UV-vis and PL spectra, and the TEM image of purified PEG₁₈-thiolate-protected CdSe NCs. The CdSe NCs displayed an UV-vis absorption maxima at 424 nm with an average size of 1.6 ± 0.3 nm. Interestingly, even though the TEM analysis showed both PEG₆- and PEG₁₈-thiolate-protected CdSe NCs were similar in size (1.6 nm in diameter), their corresponding lowest energy UV-vis absorption maxima were 430 and 424 nm, respectively. This 42 meV difference in the energy could be because of the difference in the electronic coupling between NCs, which facilitated by the “solvent-like” properties of PEGs, and we are actively pursuing this hypothesis. Nevertheless, both PEG₆- and PEG₁₈-thiolate-protected CdSe NCs were used to further investigate the influence of surface ligand chemistry on their emission properties as discussed below.

Modulating the emission properties of PEG-thiolate-protected CdSe NCs

In the case of ultrasmall (<2.0 nm diameter) NCs, most of the atoms are present on the surface. Therefore, any unpassivated surface Cd or Se sites could result in surface trap states, and the NCs could potentially display trap state emission. Depending on the physicochemical nature of the surface passivating ligands, ultrasmall CdSe NCs can display either trap state^16,17 or a combination of band-edge and trap state emission.^16,17,21 Even though the PLQY of long aliphatic ligand-protected CdSe can be as large as 10%, they are only soluble in organic solvent and incompatible with biological systems. One of the advantages of our PEG-thiolate-protected CdSe NCs is that they are soluble in aqueous medium, which is ideal for nanobiotechnology applications. However, the X-type ligand thiolate not only binds to surface Cd sites leaving Se sites unpassivated, but also significantly quenches the CdSe NCs PL properties. The quenching of PL emission is expected to be more prominent for NCs of ultra-small size, because most of their atoms are at the surface and resulting PL properties (broad deep trap) are dominated by surface occupied atoms. In this context, no PLQY of thiolate ligand-coated <2.0 nm CdSe NCs is available in the literature.^24,26,48 Therefore, an additional modification of the surface of thiolate ligand-protected NCs through appropriate surface ligand chemistry to PEG-coated NCs might produce fluorescent NCs that could be used for biological applications.

Recently it was reported that PLQY of 1.5 nm CdSe NCs passivated with mixed long aliphatic chain containing acids and amines can be enhanced up to 45% from a QY of ∼5% through post synthetic treatment with formic acid.⁶² In our initial investigation, a similar surface ligand treatment resulted in immediate aggregation of our PEG₆-thiolate-protected CdSe NCs. This could be due to the presence of formic acid in the reaction mixture that protonates the surface-attached thiolates to thiols, which would detach from the NC surface resulting in fast aggregation of unpassivated CdSe NCs.

Very recently, we have shown that passivation of surface Se sites of octylamine-protected ∼1.6 nm CdSe NCs with TPP enhanced the PL quantum yield nearly 400% (four times).⁴⁹ Through various spectroscopic analyses we determined that TPP and octylamine were attached to surface Se and Cd sites, respectively. These resulting mixed ligand-coated CdSe NCs were only soluble in organic solvents, which hindered their biological application, but such limitations should not apply to our PEG-coated NCs. Because of the restriction of TPP solubility to organic solvents, the post-synthetic surface modification of PEG_n-thiolate-protected CdSe NCs can only be performed in such solvents. Therefore, we performed the TPP treatment of PEG_n-thiolate-protected CdSe NCs in DCM (see Experimental section for details) where both NCs and TPP are completely soluble. Additionally, mixed TPP- and PEG_n-thiolate-protected CdSe NCs produced a homogenous solution, which prevented aggregation while the mixed ligand-protected NCs formed. Fig. 5A compares the UV-visible absorption spectrum of purified PEG₆-thiolate-protected CdSe NCs before and after TPP treatment. No noticeable change in the lower energy absorption peak position or shape was observed. Fig. 5B shows the PL spectra of PEG₆-thiolate-protected and TPP treated PEG₆-thiolate-protected CdSe NCs, which displayed PL-QYs of ∼0.7% and 6.3%, respectively. Although this substantial increase in PL intensity was observed after TPP treatment (Fig. 5B) no significant change in the full width at half maximum of the peak was detected.

Fig. 5 The optical properties of PEG₆-thiolate-protected CdSe NCs before (red) and after (blue) TPP treatment: (A) UV-visible absorption and (B) emission spectra. The spectra were taken by dissolving the NCs in DCM. The excitation wavelength for the PL spectra was 365 nm.

We also measured the PLQY of PEG₆-thiolate-protected ultra-small CdSe NCs after addition of TPP as a function of time, and Fig. 6 shows that the QY of the NCs increased monotonically. We hypothesize that the increase of QY is due to the stabilization of nonradiative surface trap states by passivating the surface Se sites. Importantly, within 2 h of TPP addition, the QY had increased nearly 500% with a total eight fold increase over 10 h. We believe this could be because initially all Se sites are empty and over the time some sites become capped by TPP, which increases steric hindrance for additional incoming TPP and prevents the adsorption of more ligands on neighboring surface Se sites. Furthermore, TPP binding likely reduced the flexibility of the PEG chains and hindered the TPP entering through the existing PEG-thiolate layer. Therefore, it is suggested that only a modest number of Se sites were passivated with TPP molecules, which did passivate the nonradiative trap states to a certain and increased quantum yield. The purified PEG₆-thiolate-protected CdSe NCs displayed a PL peak maximum at 547 nm in DCM and after TPP treatment the trap state emission peak of the NCs shifted to shorter wavelength by ∼3 nm over the time of our experiment. Taken together, the increase of QY and slight shift of peak position suggest that the chemical environment at the NC surface changes upon TPP addition. After the NCs displayed stable PL intensity, they were purified and no change in the PL peak position or QY was observed. Importantly, the purified NCs displayed bright yellow color upon illumination with 365 nm UV light (Fig. 7). The post-synthetic TPP treatment was also performed under identical reaction conditions on the longer PEG₁₈-thiolate-protected CdSe NCs. These NCs displayed similar PL-QY enhancement as observed for PEG₆-thiolate-protected CdSe NCs. ESI-Fig. 4† shows the PL-QY of PEG₁₈-thiolate-protected ultra-small CdSe NCs after addition of TPP as a function of time.

Fig. 6 Normalized PL-QY of PEG₆-thiolate-protected ultra-small CdSe NCs in DCM after addition of TPP relative to the initial yield (QY₀).

Fig. 7 Schematic representation of surface modification and emission property of ultra-small CdSe NCs. The Cd sites were ligated through the sulfur of the PEG-thiolate and the Se sites through the phosphorus of TPP. Both photographs of the UV cell were taken under identical illumination with a 365 nm UV light source. The PEG₆-thiolate-protected CdSe NCs displayed almost no light emission whereas after TPP treatment they displayed bright yellow color.

To test our hypothesis that TPP treatment of PEG₆-thiolate-protected ultra-small CdSe NCs would result in mixed surface passivation in which TPP was bound to surface Se sites, the TPP-treated purified sample was analyzed by X-ray photoelectron spectroscopy (XPS) (see Fig. 8 and ESI-Fig. 5†). The XPS data exhibited peaks at 129.1, 134.2 and 162.1 eV, which are due to P 2p free and CdSe surface-bound TPP, respectively. The peak at 134.2 eV is ∼5.1 eV higher than free P and this large difference is because P is datively bound to Se sites.⁶³ Since TPP forms an L-type of bond with Se atoms, it has the ability to undergo dynamic equilibrium (i.e., an adsorption–desorption process) in solution.

Fig. 8 XPS spectrum of P 2p region. Two P 2p peaks at 129.1 and 134.2 eV are observed due to free TPP and CdSe NC surface bound TPP, respectively.

Additionally, the S 2s peak at 162.3 eV was detected. The XPS data suggest that the post synthetic ligand treatment did not displace the original surface passivating ligand (PEG-thiolate) from the CdSe NC surface. Phosphorous coordinates to surface Se sites of the NCs, and therefore we would not expect detachment of thiolates because they bind strongly to Cd surface sites. Additionally, Cd 3d_5/2, Cd 3d_3/2, and Se 3d peaks were centered at 404.5, 411.4, and 53.1 eV, respectively. The sharp peak observed in the Cd 3d region and the absence of a peak at ∼60 eV indicated that the CdSe NCs did not contain CdO or SeO₂ on their surface.⁶³ These XPS data are also important in the context of the post synthetic ligand treatment and purification steps that we have adopted in our synthesis to enhance PLQY, and they suggest that our procedure is very efficient and the NCs did not undergo photooxidation.

Intracellular imaging studies using ultrasmall CdSe NCs

The majority of the literature reports describing the synthesis of ultra-small CdSe NCs employed long aliphatic hydrocarbon chain ligands to passivate the NC surface. Unfortunately, such NCs are not suitable for biological applications because of solubility issues. It was reported that QDs with <6.0 nm hydrodynamic radii have (1) low toxicity in biological systems, (2) faster diffusion into cells, and (3) faster renal clearance.⁶⁴ We used the dynamic light scattering (DLS) technique to determine the hydrodynamic radii, polydispersity index, and zeta-potential for PEG₆- and PEG₁₈-thiolate-protected ultra-small CdSe NCs (see ESI-Table 1†). The hydrodynamic radii of PEG₆- and PEG₁₈-thiolate-protected ultra-small CdSe NCs were found to be 4.3 ± 6.4 and 4.5 ± 4.4 nm, respectively. TEM analysis showed that the CdSe NC core radius was 0.8 nm, whereas fully stretched (ChemBioDraw 13.0) PEG₆- and PEG₁₈-thiols lengths are 2.2 and 6.2 nm, respectively, therefore hydrodynamic radii would be approximately 3.0 and 7.0 nm, respectively. The experimental value for the radii (4.3 nm) of PEG₆-thiolate-protected CdSe NCs was slightly larger than the calculated value of 3.0 nm, which suggested that some aggregation occurred when the NCs were dissolved in solution. Interestingly, 4.5 nm hydrodynamic radii was determined for PEG₁₈-thiolate-protected CdSe NCs, which is lower than the calculated value of 7.0 nm, indicating that the PEG₁₈ chains were not fully extended in the solution. Nevertheless, these values are much lower than recently reported QDs of ∼12 nm which caused increased cytotoxicity and slower renal clearance.⁶⁴ Finally, the zeta-potential of PEG₆- and PEG₁₈-thiolate-protected CdSe NCs were −19.2 and −32.0 mV, respectively. These values suggest that PEG₁₈-thiolate-protected CdSe NCs were more stable in solution than PEG₆-thiolate-protected ones.

Our TPP-treated PEG_n-thiolate-protected <2.0 nm CdSe NCs are not only soluble in an aqueous medium but are also expected to have all the above-mentioned beneficial characteristics. Furthermore, it is also known that passivated QDs with PEGylated ligands can reduce nonspecific interactions.⁶⁵ In our initial in vitro investigation, the shorter chain length (PEG₆) thiolate ligand-protected bright yellow CdSe NCs were used for imaging studies. We used green fluorescent protein-transfected fibroblast cells, which displayed green PL highlighting cellular structures upon light excitation. Differential interference contrast (DIC) micrographs showed that the NCs aggregated around the cell membranes (ESI-Fig. 6†) and no NC fluorescence emission was observed outlining specific internal cellular structures. This could be because the high hydrophobicity of TPP did not allow NC penetration through the cell membranes even though the NC surface was coated with PEG-thiolate ligands. The small fraction of NCs entered inside the cell lost their fluorescent properties either due to aggregation or detachment of TPP during the diffusion through cell membranes. TPP extends out from the NP surface nearly 0.6 nm (ChemBioDraw 3D) and the PEG₆ shell thickness is approximately 0.4 nm.^66,67 Thus, the hydrophobic character of TPP dominated the hydrophilic character of the PEG-thiolate resulting in a high probability of TPP detachment.

In order to further investigate potential cell membrane penetration, the longer chain length PEG₁₈-thiolate was used to overcome the hydrophobic character imposed by TPP on the mixed ligand-coated CdSe NCs. The PL properties of TPP-treated PEG₁₈-thiolate-protected CdSe NCs exhibited the same QY as those coated with PEG₆ thiolate (ESI-Fig. 4†). Thus we investigated the cellular uptake ability of the fibroblast cells for TPP-treated PEG₁₈-thiolate-protected CdSe NCs. Fig. 9A illustrates the DIC image in which NCs were asymmetrically aggregated around the cell nucleus as reported by others in the literature.^68,69 However, individual non aggregated CdSe NCs inside the cell were not observed in the DIC analysis due to the limitations in magnification and resolution of the confocal microscope. Nevertheless, the fluorescence image (Fig. 9C) shows that the CdSe NCs were well dispersed inside the fibroblast cell. We also used these transfected cells to monitor survival for a period of up to 5 days in the presence of 100 nM NCs. Most importantly, the fluorescence properties of TPP-treated PEG₁₈-thiolate-protected CdSe NCs inside the cell did not degrade for a period of five days. The green fluorescent protein-transfected fibroblast cells after incubated with mixed TPP- and PEG₁₈-thiolate-protected CdSe NCs for five days were further incubated with 200 μL of LIVE/DEAD viability stock solution (Invitrogen Co, USA) for 40 min at 30 °C. The cells were then rinsed with PBS buffer followed by imaging with a Confocal EPI microscopy. The green and red fluorescent cells, which represent live and dead cells, were then counted. These experimental data are very significant and suggest that more than 95% cells were alive upon incubation with our mixed ligand-protected CdSe NCs even after 5 days of incubation. This result is important in the context of the enhanced stability of CdSe NCs versus other fluorescent labels as well as the specific stability of the metal–thiolate bond inside the cell. We believe two factors played an important role in the cell survival when the PEG₁₈-thiolate-protected CdSe NCs were used for intracellular imaging studies: (1) the moderately long chain PEG-thiolate coating was able to reduce the cytotoxicity of CdSe NCs by preventing leakage of toxic metal ions (i.e., Cd²⁺), and (2) only ∼34 Cd are present per NCs reducing leakage of ions into their cellular surroundings in comparison with traditionally used QDs which contained hundreds of metal ions in their core.

Fig. 9 (A) Representative DIC image of a green fluorescence protein transfected fibroblast cell incubated with ∼100 nM TPP-treated PEG₁₈-thiolate-protected CdSe NCs as described in the experimental section, (B) merged NCs and green fluorescence protein transfected cell (C) NCs with green fluorescent background removed, and (D) transfected cell before NC treatment. The inserts in B–D show expanded regions of the cell as marked by the white square box. Close examination of the fluorescence in C shows that the NCs penetrate the outer cell membranes, but not the nuclear membrane, which totally lacks fluorescence from NC. The NCs are red not yellow because of the wavelength filter used. A coverslip was placed on top of the disk to restrict the cell movement during the addition of the CdSe NC solution.

Mixed PEG₁₈-thiolate and TPP-coated CdSe NCs were dissolved in water and exposed to hand-held UV light (365 nm wavelength) for five minutes and the absorption spectrum was collected showing no change. The same experiment was conducted on the same NC solution for a period of 24 hours and no noticeable change in the absorption peak intensity was observed (see ESI-Fig. 9B†). These data clearly indicate that our mixed ligand-coated NCs are highly stable under substantial UV exposure.

Attaching organic fluorophores or fluorescent proteins to biomolecules is commonly used for cellular imaging.^70,71 These organic dyes and fluorescent proteins have narrow excitation spectral windows and generally undergo fast chemical and photodegradation, which limits their applications in biotechnology. All the above-mentioned drawbacks have been substantially eliminated by using QDs.^7,45,72,73 However, cytotoxicity is a major challenge in the application of QDs in nanobiotechnology. Here we have shown that ultrasmall CdSe NCs can be applied to cellular imaging by enhancing their physicochemical and photophysical properties, and they could be an alternative for organic fluorophores, fluorescent proteins, or QDs. In order to address the challenges associated with the dosage-dependent cytotoxicity effects in the present system the following experiments were conducted. Briefly, plated fibroblast cells were incubated at different concentrations ranging from 500 nM to 100 μM of mixed PEG₁₈-thiolate and TPP-coated CdSe NCs for 24 h, rinsed with PBS buffer and then applied LIVE/DEAD viability stock solution for 40 min before imaging. Fig. 10 illustrates the cell survival over different NC concentration, which showed >95% of the cells were alive. Additionally, to proceed to whole animal studies with PEG₁₈-thiolate-protected CdSe NCs some important physicochemical and photophysical properties require further investigation and perhaps improvement: (1) the long-term stability of mixed ligand-coated NCs in wide range of pH and electrolyte solutions, (2) the extent of non-specific interactions with proteins and smaller molecules in physiological media (e.g., plasma or serum), and (3) further PL-QY enhancement through suitable surface ligand chemistry.

Fig. 10 (A) Fluorescence image of the fibroblast cells after 24 h of 500 nM of mixed TPP- and PEG₁₈-thiolate-coated CdSe NCs incubation followed by rinsing with PBS and applied LIVE/DEAD viability stock solution for 40 min. (B) The LIVE/DEAD assay on fibroblast cells incubated to different concentration of PEG₁₈-thiolate-coated CdSe NCs for 24 h. 200 cells were counted to determine the cell viability. The green channel monitors healthy cells while the red represents cellular death.

Conclusion

In summary, a simple synthetic method and single step purification procedure has been developed for the synthesis of PEG_n-thiolated-protected ultrasmall CdSe NCs. Along with optical spectroscopy, high-resolution MALDI-TOF-MS analysis has demonstrated the formation of a stoichiometry core of possible composition (CdSe)_33/34. These PEG_n-thiolate-protected CdSe NCs showed unusual solubility properties, being readily soluble in a wide array of organic solvents including CH₃CN, CH₂Cl₂, EtOH, and CHCl₃. This unique solubility allowed us to prepare bright yellow light emitting CdSe NCs through a post synthetic ligand treatment with TPP on PEG_n-thiolated-protected NCs. The XPS analysis unequivocally proved that the NC surface is coated with TPP and PEG-thiolate ligands. TPP preferentially bound to the surface Se sites through a dative bond, which reduced the formation of nonradiative trap state formation and enhanced the PL-QY. Because of their aqueous solubility and bright emission properties, the mixed ligand-coated ultra-small CdSe NCs were used in in vitro cell imaging. The observed cellular viability for at least five days after treatment with CdSe NCs suggests potential use in live animal studies, in which their small size should increase the rate of clearance of the NCs from the body.⁶⁴

Acknowledgements

This work was supported by Start-up funds provided to R. S. by IUPUI. We also like to thank Prof. Barry Muhoberac for helpful discussion, Dr Carrie Donley (UNC-Chapel Hill) for assistance with XPS analysis, and Dr Merrell Johnson for helping with dynamic light scattering measurement. The Bruker 500 MHz NMR was purchased using funds from an NSF-MRI award (CHE-0619254). A. D. gratefully acknowledges support from NSF 0903787. S. D. thanks INDI-IUPUI for financial support.

References

1. M. L. Steigerwald and L. E. Brus, Acc. Chem. Res., 1990, 23, 183–188.
2. J. H. Bang and P. V. Kamat, ACS Nano, 2009, 3, 1467–1476.
3. J. H. Yu, X. Liu, K. E. Kweon, J. Joo, J. Park, K.-T. Ko, D. W. Lee, S. Shen, K. Tivakornsasithorn, J. S. Son, J.-H. Park, Y.-W. Kim, G. S. Hwang, M. Dobrowolska, J. K. Furdyna and T. Hyeon, Nat. Mater., 2010, 9, 47–53.
4. J.-S. Lee, M. V. Kovalenko, J. Huang, D. S. Chung and D. V. Talapin, Nat. Nanotechnol., 2011, 6, 348–352.
5. K. Tvrdy, P. A. Frantsuzov and P. V. Kamat, Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 29–34.
6. A. P. Alivisatos, Science, 1996, 271, 933–937.
7. M. Bruchez, M. Moronne, P. Gin, S. Weiss and A. P. Alivisatos, Science, 1998, 281, 2013–2016.
8. V. I. Klimov, A. A. Mikhailovsky, S. Xu, A. Malko, J. A. Hollingsworth, C. A. Leatherdale, H.-J. Eisler and M. G. Bawendi, Science, 2000, 290, 314–317.
9. C. B. Murray, C. R. Kagan and M. G. Bawendi, Science, 1995, 270, 1335–1338.
10. I. n. Robel, M. Kuno and P. V. Kamat, J. Am. Chem. Soc., 2007, 129, 4136–4137.
11. M. C. Schlamp, X. Peng and A. P. Alivisatos, J. Appl. Phys., 1997, 82, 5837–5842.
12. D. Pacifici, H. J. Lezec and H. A. Atwater, Nat. Photonics, 2007, 1, 402–406.
13. V. L. Colvin, M. C. Schlamp and A. P. Alivisatos, Nature, 1994, 370, 354–357.
14. D. C. Oertel, M. G. Bawendi, A. C. Arango and V. Bulovic, Appl. Phys. Lett., 2005, 87, 213505.
15. M. Drndic, M. V. Jarosz, N. Y. Morgan, M. A. Kastner and M. G. Bawendi, J. Appl. Phys., 2002, 92, 7498–7503.
16. B. M. Cossairt and J. S. Owen, Chem. Mater., 2011, 23, 3114–3119.
17. J. C. Newton, K. Ramasamy, M. Mandal, G. K. Joshi, A. Kumbhar and R. Sardar, J. Phys. Chem. C, 2012, 116, 4380–4389.
18. B. M. Cossairt, P. Juhas, S. J. L. Billinge and J. S. Owen, J. Phys. Chem. Lett., 2011, 2, 3075–3080.
19. A. D. Dukes, J. R. McBride and S. J. Rosenthal, Chem. Mater., 2010, 22, 6402–6408.
20. M. Zanella, A. Z. Abbasi, A. K. Schaper and W. J. Parak, J. Phys. Chem. C, 2010, 114, 6205–6215.
21. M. J. Bowers, J. R. McBride and S. J. Rosenthal, J. Am. Chem. Soc., 2005, 127, 15378–15379.
22. F. S. Riehle, R. Bienert, R. Thomann, G. A. Urban and M. Kruger, Nano Lett., 2009, 9, 514–518.
23. E. Kuçur, J. Ziegler and T. Nann, Small, 2008, 4, 883–887.
24. Y.-S. Park, A. Dmytruk, I. Dmitruk, A. Kasuya, M. Takeda, N. Ohuchi, Y. Okamoto, N. Kaji, M. Tokeshi and Y. Baba, ACS Nano, 2009, 4, 121–128.
25. S. Kudera, M. Zanella, C. Giannini, A. Rizzo, Y. Li, G. Gigli, R. Cingolani, G. Ciccarella, W. Spahl, W. J. Parak and L. Manna, Adv. Mater., 2007, 19, 548–552.
26. Y.-S. Park, A. Dmytruk, I. Dmitruk, A. Kasuya, Y. Okamoto, N. Kaji, M. Tokeshi and Y. Baba, J. Phys. Chem. C, 2010, 114, 18834–18840.
27. Q. Yu and C.-Y. Liu, J. Phys. Chem. C, 2009, 113, 12766–12771.
28. C. M. Evans, A. M. Love and E. A. Weiss, J. Am. Chem. Soc., 2012, 134, 17298–17305.
29. C. Landes, M. Braun, C. Burda and M. A. El-Sayed, Nano Lett., 2001, 1, 667–670.
30. C. M. Evans, L. Guo, J. J. Peterson, S. Maccagnano-Zacher and T. D. Krauss, Nano Lett., 2008, 8, 2896–2899.
31. G. D. Lilly, A. C. Whalley, S. Grunder, C. Valente, M. T. Frederick, J. F. Stoddart and E. A. Weiss, J. Mater. Chem., 2011, 21, 11492–11497.
32. E. E. Lees, T.-L. Nguyen, A. H. A. Clayton and P. Mulvaney, ACS Nano, 2009, 3, 1121–1128.
33. J. T. Siy, E. M. Brauser and M. H. Bartl, Chem. Commun., 2011, 47, 364–366.
34. D. V. Talapin, J.-S. Lee, M. V. Kovalenko and E. V. Shevchenko, Chem. Rev., 2009, 110, 389–458.
35. K. Yu, M. Z. Hu, R. Wang, M. L. Piolet, M. Frotey, M. B. Zaman, X. Wu, D. M. Leek, Y. Tao, D. Wilkinson and C. Li, J. Phys. Chem. C, 2010, 114, 3329–3339.
36. A. J. Morris-Cohen, M. T. Frederick, G. D. Lilly, E. A. McArthur and E. A. Weiss, J. Phys. Chem. Lett., 2010, 1, 1078–1081.
37. Y. C. Cao and J. Wang, J. Am. Chem. Soc., 2004, 126, 14336–14337.
38. Z. A. Peng and X. Peng, J. Am. Chem. Soc., 2000, 123, 183–184.
39. F. Wang, R. Tang, J. L. F. Kao, S. D. Dingman and W. E. Buhro, J. Am. Chem. Soc., 2009, 131, 4983–4994.
40. D. V. Talapin, A. L. Rogach, A. Kornowski, M. Haase and H. Weller, Nano Lett., 2001, 1, 207–211.
41. M. A. Schreuder, K. Xiao, I. N. Ivanov, S. M. Weiss and S. J. Rosenthal, Nano Lett., 2010, 10, 573–576.
42. H. Kloust, C. Schmidtke, J.-P. Merkl, A. Feld, T. Schotten, U. E. A. Fittschen, M. Gehring, J. Ostermann, E. Poselt and H. Weller, J. Phys. Chem. C, 2013, 117, 23244–23250.
43. W. W. Yu, E. Chang, J. C. Falkner, J. Zhang, A. M. Al-Somali, C. M. Sayes, J. Johns, R. Drezek and V. L. Colvin, J. Am. Chem. Soc., 2007, 129, 2871–2879.
44. O. Carion, B. Mahler, T. Pons and B. Dubertret, Nat. Protoc., 2007, 2, 2383–2390.
45. I. L. Medintz, H. T. Uyeda, E. R. Goldman and H. Mattoussi, Nat. Mater., 2005, 4, 435–446.
46. A. Chakraborty, A. R. Maity and N. R. Jana, RSC Adv., 2014, 4, 10434–10438.
47. S. F. Wuister, C. de Mello Donegá and A. Meijerink, J. Phys. Chem. B, 2004, 108, 17393–17397.
48. A. L. Rogach, A. Kornowski, M. Gao, A. Eychmueller and H. Weller, J. Phys. Chem. B, 1999, 103, 3065–3069.
49. S. Dolai, P. R. Nimmala, M. Mandal, B. B. Muhoberac, K. Dria, A. Dass and R. Sardar, Chem. Mater., 2014, 26, 1278–1285.
50. S. M. Harrell, J. R. McBride and S. J. Rosenthal, Chem. Mater., 2013, 25, 1199–1210.
51. A. Kasuya, R. Sivamohan, Y. A. Barnakov, I. M. Dmitruk, T. Nirasawa, V. R. Romanyuk, V. Kumar, S. V. Mamykin, K. Tohji, B. Jeyadevan, K. Shinoda, T. Kudo, O. Terasaki, Z. Liu, R. V. Belosludov, V. Sundararajan and Y. Kawazoe, Nat. Mater., 2004, 3, 99–102.
52. A. W. Snow and E. E. Foos, Synthesis, 2003, 4, 509–512.
53. S. Senthilkumar, S. Nath and H. Pal, Photochem. Photobiol., 2004, 80, 104–111.
54. J. Jasieniak, L. Smith, J. v. Embden, P. Mulvaney and M. Califano, J. Phys. Chem. C, 2009, 113, 19468–19474.
55. R. Jose, Z. Zhelev, R. Bakalova, Y. Baba and M. Ishikawa, Appl. Phys. Lett., 2006, 89, 013115–013113.
56. A. Badia, L. Demers, L. Dickinson, F. G. Morin, R. B. Lennox and L. Reven, J. Am. Chem. Soc., 1997, 119, 11104–11105.
57. M. J. Hostetler, J. E. Wingate, C.-J. Zhong, J. E. Harris, R. W. Vachet, M. R. Clark, J. D. Londono, S. J. Green, J. J. Stokes, G. D. Wignall, G. L. Glish, M. D. Porter, N. D. Evans and R. W. Murray, Langmuir, 1998, 14, 17–30.
58. X. Ji, D. Copenhaver, C. Sichmeller and X. Peng, J. Am. Chem. Soc., 2008, 130, 5726–5735.
59. A. Dass, J. Am. Chem. Soc., 2009, 131, 11666–11667.
60. A. Dass, A. Stevenson, G. R. Dubay, J. B. Tracy and R. W. Murray, J. Am. Chem. Soc., 2008, 130, 5940–5946.
61. N. C. Anderson, M. P. Hendricks, J. J. Choi and J. S. Owen, J. Am. Chem. Soc., 2013, 135, 18536–18548.
62. T. E. Rosson, S. M. Claiborne, J. R. McBride, B. S. Stratton and S. J. Rosenthal, J. Am. Chem. Soc., 2012, 134, 8006–8009.
63. J. E. B. Katari, V. L. Colvin and A. P. Alivisatos, J. Phys. Chem., 1994, 98, 4109–4117.
64. H. Soo Choi, W. Liu, P. Misra, E. Tanaka, J. P. Zimmer, B. Itty Ipe, M. G. Bawendi and J. V. Frangioni, Nat. Biotechnol., 2007, 25, 1165–1170.
65. E. L. Bentzen, I. D. Tomlinson, J. Mason, P. Gresch, M. R. Warnement, D. Wright, E. Sanders-Bush, R. Blakely and S. J. Rosenthal, Bioconjugate Chem., 2005, 16, 1488–1494.
66. M. E. Williams and R. W. Murray, Chem. Mater., 1998, 10, 3603–3610.
67. M. E. Williams, H. Masui, J. W. Long, J. Malik and R. W. Murray, J. Am. Chem. Soc., 1997, 119, 1997–2005.
68. M. H. Stewart, K. Susumu, B. C. Mei, I. L. Medintz, J. B. Delehanty, J. B. Blanco-Canosa, P. E. Dawson and H. Mattoussi, J. Am. Chem. Soc., 2010, 132, 9804–9813.
69. H. T. Uyeda, I. L. Medintz, J. K. Jaiswal, S. M. Simon and H. Mattoussi, J. Am. Chem. Soc., 2005, 127, 3870–3878.
70. T. Ried, A. Baldini, T. C. Rand and D. C. Ward, Proc. Natl. Acad. Sci. U. S. A., 1992, 89, 1388–1392.
71. E. Schrock, S. d. Manoir, T. Veldman, B. Schoell, J. Wienberg, M. A. Ferguson-Smith, Y. Ning, D. H. Ledbetter, I. Bar-Am, D. Soenksen, Y. Garini and T. Ried, Science, 1996, 273, 494–497.
72. W. C. W. Chan and S. Nie, Science, 1998, 281, 2016–2018.
73. X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir and S. Weiss, Science, 2005, 307, 538–544.

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Footnote

† Electronic supplementary information (ESI) available: Synthesis of PEG_n-thiols, additional absorption, emission, XRD, and XPS spectra, and TEM and cell images. See DOI: 10.1039/c4ra02549k

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