Purple-, Blue-, and Green-Emitting Multishell Alloyed Quantum Dots

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Purple‑, Blue‑, and Green-Emitting Multishell Alloyed Quantum Dots: Synthesis, Characterization, and Application for Ratiometric Extracellular pH Sensing Kimihiro Susumu,*,†,§ Lauren D. Field,‡,∥ Eunkeu Oh,†,§ Michael Hunt,† James B. Delehanty,‡ Valle Palomo,⊥ Philip E. Dawson,⊥ Alan L. Huston,† and Igor L. Medintz*,‡ †

Optical Sciences Division, Code 5600, ‡Center for Bio/Molecular Science and Engineering, Code 6900, United States Naval Research Laboratory, Washington, D.C. 20375, United States § Sotera Defense Solutions, Columbia, Maryland 21046, United States ∥ Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States ⊥ Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, United States S Supporting Information *

ABSTRACT: We report the synthesis of a series of CdxZn1−xSe/ CdyZn1−yS/ZnS and ZnSe/CdyZn1−yS/ZnS multishell alloyed luminescent semiconductor quantum dots (QDs) with fluorescence maxima ranging from 410 to 530 nm which cover the purple, blue, and green portion of the spectrum. Their subsequent surface modification to prepare water-soluble blue-emitting QDs, characterization, and application for ratiometric pH sensing in aqueous buffers and in an extracellular environment are further described. QDs were synthesized starting from ZnSe cores, and the fluorescence peak positions were tuned by (i) cation exchange with cadmium ions and/ or (ii) overcoating with CdyZn1−yS layers. The as-prepared QDs had reasonably high fluorescence quantum yields (∼30−55%), narrow fluorescence bands (fwhm ∼25−35 nm), and monodispersed semispherical shapes. Ligand exchange with hydrophilic compact ligands was successfully carried out to prepare a series of water-soluble blue-emitting QDs. QDs coated with the hydrophilic compact ligands preserved the intrinsic photophysical properties well and showed excellent colloidal stability in aqueous buffers for over a year. The blue-emitting QDs were further conjugated with the pH-sensitive dye, fluorescein isothiocyanate (FITC), to construct a fluorescence resonance energy transfer-based ratiometric pH sensing platform, and pH monitoring with the QD-FITC conjugates was successfully demonstrated at pHs ranging between 3 and 7.5. Further assembly of the QD-FITC conjugates with membrane localization peptides allowed monitoring of the pH in extracellular environments. High quality, water-soluble blue-emitting QDs coated with compact ligands can help expand the practical fluorescence range of QDs for a variety of biological applications.

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fluorescence bands and reasonable air stability. Synthetic methods for QDs with fluorescence in the visible region from green to red colors have already been well-established using a series of size-tuned CdSe/ZnS and CdSe/CdS core/shell QDs.8−10 Synthesis of blue-emitting CdSe/ZnS QDs has also been reported.11,12 However, the utility of blue-emitting CdSe/ ZnS QDs has not been fully demonstrated because the intrinsic material properties of CdSe are not ideal to cover the entire blue color range without compromising the material quality, including color purity, QYs, and stability. Moreover, synthesis of small CdSe cores of high quality and their subsequent overcoating is still quite challenging. To prepare blue-emitting CdSe/ZnS QDs, the initial CdSe core size has to be extremely

INTRODUCTION

Since the facile synthesis of colloidal cadmium chalcogenide quantum dots (QDs) was first reported,1 QDs have attracted a great deal of attention over conventional organic fluorophores due to their unique photophysical properties, including size dependent fluorescence spectra, broad excitation spectra, high molar absorptivity, and photochemical stability.2 Their potential applications span many disparate areas ranging from light emitting diodes (LEDs), solar cells, and catalysts to sensors and biomedical imaging.3−6 For many of these applications, the critical importance of the QD core/shell structure has been repeatedly demonstrated as a key prerequisite to maintaining high fluorescence quantum yields (QYs) and to enhancing long-term photochemical stability.7 CdSe is one of the most popular binary semiconducting materials studied for synthesizing QDs due to the wide spectral coverage it affords across the visible region along with narrow © 2017 American Chemical Society

Received: May 30, 2017 Revised: August 4, 2017 Published: August 30, 2017 7330

DOI: 10.1021/acs.chemmater.7b02174 Chem. Mater. 2017, 29, 7330−7344

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Chemistry of Materials

Scheme 1. Synthetic Scheme of Multishell QDs Starting from ZnSe Core via (i) Cation Exchange with Cd2+, (ii) Overcoating with CdyZn1−yS Layers, and (iii) Overcoating with ZnS Layers.a

a

(A) CdxZn1−xSe/CdyZn1−yS/ZnS and (B) ZnSe/CdyZn1−yS/ZnS. It should be noted that the multilayer QD compositions, including Cd:Zn ratio, are described based on the initial precursor combinations and ratios used for the QD synthesis throughout this study; the actual core/shell structure and element ratios in the final product would be most likely different due to alloy formation.

from the violet to the red region of the spectrum depending on composition ratio and QD size. In particular, systematic tuning of Cd/Zn ratios in CdxZn1−xSe QDs can be achieved via cation exchange of ZnSe QDs with Cd2+ ions.26,27 Increasing the amount of Cd2+ ions used during cation exchange with ZnSe core QDs progressively causes a red shift of the fluorescence band, making it somewhat facile to tune the fluorescence colors of ternary CdxZn1−xSe QDs from violet to blue and then green. Similar facile color tuning has not been fully established for CdSexS1−x ternary QDs. In addition, recent studies have demonstrated high QYs and robustness of CdxZn1−xS/ZnS ternary core/shell QDs as promising blue emitting materials.28−31 The fluorescence peaks of CdxZn1−xS/ZnS QDs have currently reached up to only ∼475 nm. Thus, CdxZn1−xSe remains a promising material to develop highly luminescent QDs with potential fluorescence peaks in the deep blue to green target range. While a number of studies have reported synthetic methods for blue-emitting QDs, most have not demonstrated actual utility beyond fabrication of LEDs.29,30,32 Applications for biological sensing and imaging require further surface modification of the QDs to make them soluble in aqueous media without compromising their inherent photophysical properties even under harsh biological environments that can be acidic or high in ionic content. Interestingly, most of the limited examples of water-soluble blue-emitting QDs have been prepared by encapsulation techniques33−37 with only a few studies reporting preliminary demonstrations of direct ligand exchange of blue-emitting QDs.31,38 Ligand exchange of hydrophobic QDs requires the original hydrophobic surface ligands to be stripped off and then replaced with hydrophilic

small (∼2 nm or less), which is not a trivial task especially when trying to also attain narrow size distributions. Small QD cores are also less emissive and far less stable than the larger ones due to the larger surface-to-volume ratios. Therefore, it is almost a necessity to overcoat the small CdSe cores with higher band gap materials such as ZnS for practical use. However, such overcoating usually results in a progressive red-shift of the fluorescence peak during shell growth, and the QD emission can quickly go beyond 500 nm even with minimum layering of ZnS shell. Overcoating of small CdSe cores with a thin ZnS shell is also usually not sufficient to maintain high fluorescence QYs and long-term colloidal stability. Thus, it is quite challenging to keep the fluorescence colors of CdSe/ZnS core/shell QDs in the blue region without compromising crucial material requirements. The other common binary semiconducting materials that have been explored for preparing violet-to-blue emitting QDs are ZnSe and CdS. However, the fluorescence peaks of ZnSe and CdS QDs normally do not reach beyond ∼460 and ∼480 nm, respectively,13−17 and can sometimes be compromised by red-shifted defect emissions. Combined, this has limited the ability to prepare QD materials that are emission-tuned from the blue to green wavelengths from a common binary semiconductor material in a facile and systematic manner. To fill the gap between blue and green emission colors and establish continuous color tuning in this entire range, CdxZn1−xSe and CdSexS1−x ternary alloy QD compositions have been studied.18−25 Here, the subscripts x and 1−x represent the molar fraction of each element in the alloy structure. It has been demonstrated that the fluorescence of CdxZn1−xSe and CdSexS1−x ternary alloyed QDs can be tuned 7331

DOI: 10.1021/acs.chemmater.7b02174 Chem. Mater. 2017, 29, 7330−7344

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Chemistry of Materials

(Malvern Instruments Ltd., Worcestershire, UK) and analyzed using Dispersion Technology Software (DTS, Malvern Instruments Ltd.). All samples were prepared in water containing 10 μM NaOH (pH 9). The QD solutions (∼50 nM) were filtered through 0.02 or 0.1 μm syringe filters (Whatman) and loaded into disposable cells, and the data were collected at 25 °C. For each sample, the autocorrelation function was the average of three runs of 10 s each, and each set was repeated three to six times. CONTIN analysis was then used to obtain number or intensity profile versus hydrodynamic size profiles for the dispersions studied.49 Structural characterization of as-prepared QDs was carried out using a JEOL 2200-FX analytical high-resolution transmission electron microscope (HR-TEM) with a 200 kV accelerating voltage. Washed QD solutions were filtered using 0.25 μm Millipore syringe filters. A drop (5−10 μL) of the filtered QD solution was spread onto ultrathin carbon/holey support film on a 300 mesh Au grid (Ted Pella, Inc.) and dried. Individual particle sizes were measured using a Gatan Digital Micrograph (Gatan, Inc., Pleasanton, CA); average sizes along with standard deviations were extracted from analysis of ∼100 nanoparticles.50 For X-ray diffraction measurements, QDs suspended in methylene chloride were placed on a specialty powder sample holder. Additional methylene chloride was used as necessary to help the QDs fill the shallow cavity on the sample holder. Samples were allowed to dry before measurements were performed using a Rigaku Smart Lab X-ray diffractometer with mirror optics to provide a parallel beam path. Analysis of each sample was performed using Rigaku’s PDXL software. Details of energy dispersive X-ray (EDX) spectroscopic measurements are available in the Supporting Information. Preparation of Precursors for QD Synthesis. TOP:Se Solution (0.5 M). Se (0.592 g, 7.5 mmol) and TOP (15.0 mL) were mixed in a 20 mL vial which was sealed with a septum. The mixture was degassed under vacuum at 80 °C for 10 min, filled with N2, and stirred until the Se was dissolved. Cd Oleate Solution (0.2 M). CdO (1.284 g, 10 mmol), oleic acid (12.69 mL, 40 mmol), and ODE (29.45 mL) were loaded into a 100 mL three-neck round-bottom flask. The mixture was degassed under vacuum at 100 °C to remove volatiles, filled with N2, heated to 240 °C to dissolve the Cd precursor, and cooled to room temperature. After oleylamine (6.58 mL, 20 mmol) was added, the mixture was degassed under vacuum at 100 °C for 30 min, backfilled with N2, and cooled to room temperature. Zn Oleate Solution (0.2 M). Zn(OAc)2·2H2O (2.634 g, 12.0 mmol), oleic acid (7.57 mL, 24 mmol), and ODE (43.8 mL) were loaded into a 100 mL three-neck round-bottom flask. The mixture was degassed under vacuum at 100 °C to remove volatiles, filled with N2, heated to 230 °C to dissolve the Zn precursor, and cooled to room temperature. After oleylamine (7.90 mL, 24 mmol) was added, the mixture was degassed under vacuum at 100 °C for 30 min, backfilled with N2, and cooled to room temperature. n-Octanethiol Solution (0.2 M). n-Octanethiol (0.52 mL, 3.0 mmol) was dissolved in 14.48 mL of ODE. ZnSe Core Synthesis. Spherical ZnSe cores were synthesized following literature procedures with some modifications.13,27,51,52 Oleylamine (20 mL) was loaded into a 100 mL four-neck roundbottom flask. The oleylamine solution was degassed under vacuum at 100 °C for 30 min. After filling with N2, the solution was heated to 295 °C, and 0.5 M TOP:Se solution (3.0 mL, 1.5 mmol) was slowly injected into the oleylamine solution. Then, 0.5 M ZnEt2 in TOP (3.75 mL, 1.875 mmol) was swiftly injected at 295 °C with vigorous stirring. The reaction mixture was kept at 290 °C, and aliquots were periodically taken to monitor core growth by absorption spectra. To grow larger cores, 0.5 M ZnEt2 in TOP and 0.5 M TOP:Se were further added dropwise via syringe pump until the lowest absorption band reached the desired wavelength. After the reaction mixture cooled to room temperature, n-butanol (14 mL) was added, and the reaction mixture was aliquoted to 40 mL vials. Excess isopropanol and ethanol were added to each vial to flocculate the QDs, and the mixtures were centrifuged at 3800 rpm for 5 min. The supernatant was discarded, and the QD pellets were dissolved in a minimum amount of toluene. This cleaning procedure was repeated once more. QD pellets

stabilizing ligands. This method can be far harsher than encapsulation techniques which preserve the original hydrophobic ligands and overcoat them with interdigitating amphiphilic polymers or lipids.39,40 While ligand exchange can often result in a sizable decrease of a QD QY, this approach significantly minimizes the hydrodynamic size of the final hydrophilic QD, which is something that has proven to be beneficial for biological sensing and imaging studies. To provide blue-emitting QDs with high QYs, color purity and colloidal stability in biological environments even after performing ligand exchange, it is essential that the native cores be effectively protected and insulated with robust shells. In this study, we focused on the visible fluorescence range below 500 nm for QD materials and synthesized a series of CdxZn1−xSe/CdyZn1−yS/ZnS and ZnSe/CdyZn1−yS/ZnS core/ multishells QDs with fluorescence ranging from purple and blue to the green region of the spectrum (∼410−530 nm). QD synthesis was started from ZnSe cores, and the fluorescence wavelengths were tuned by cation exchange reactions with Cd2+ ions and/or an interfacial alloying process during the CdyZn1−yS overcoating steps (see Scheme 1). The as-prepared hydrophobic blue-emitting QDs were further modified by ligand exchange with compact hydrophilic ligands and transformed to water-soluble QDs to evaluate the utility for biological assays because blue-emitting QDs are the least developed for biological sensing and imaging studies among QDs with visible fluorescence. Water-soluble blue-emitting QDs prepared in this study exhibited reasonably high QYs, narrow fluorescence bands, and excellent pH and salt stability. These nanocrystals were further conjugated with pH sensitive fluorescein derivatives to construct nanoscale ratiometric pH sensors. The robustness of the sensors allowed us to monitor the pH changes localized in extracellular environments.

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EXPERIMENTAL SECTION

Chemicals. Diethylzinc (ZnEt2; min. 95%), selenium (Se; 99.99%), tri-n-octylphosphine (TOP; min. 97%), and CdO were purchased from Strem Chemicals. Oleylamine (technical grade, 70%), noctanethiol (n-C8SH), and oleic acid (technical grade, 90%) were purchased from Sigma-Aldrich. 1-Octadecene (ODE; technical grade, 90%) was purchased from Acros Organics. 1-Dodecylphosphonic acid and zinc acetate dihydrate (Zn(OAc)2·2H2O) were purchased from Alfa Aesar. Fluorescein-5-isothiocyanate (FITC ’Isomer I’) was purchased from Thermo Fisher Scientific. Hydrophilic surface ligands DHLA-PEG750-OMe,41,42 DHLA-PEG600-NH2,43,44 CL2,45 and CL445 were synthesized as previously described. All other chemicals, including solvents, were purchased from Sigma-Aldrich or Acros Organics and were used as received. Instrumentation. Electronic absorption spectra were recorded using a Shimadzu UV-1800 UV−vis spectrophotometer. Fluorescence spectra were collected using a Spex Fluorolog-3 spectrophotometer (Jobin Yvon Inc., Edison, NJ) equipped with a red-sensitive R2658 Hamamatsu PMT detector or a Tecan Infinite M1000 Dual Monochromator Multifunction Microtiter Plate Reader (Tecan, Research Triangle Park, NC) with 96-well black microtiter plates (100 μL per well). QYs were measured at room temperature with quinine sulfate in 1.0 N H2SO4 (λmax = 453 nm ; Φf = 0.546)46 or coumarin 480 (coumarin 102) in ethanol (λmax = 469 nm; Φf = 0.764)47 as standards. The obtained fluorescence spectra were corrected using the spectral output of a calibrated light source supplied by the National Bureau of Standards. The concentrations of ZnSe QDs were estimated by inductively coupled plasma optical emission spectroscopy (ICP-OES) using a PerkinElmer 5300 ICPOES after acid digestion of the QD samples.48 Dynamic light scattering (DLS) measurements were carried out using ZetaSizer NanoSeries equipped with a He−Ne laser source (λ = 633 nm) 7332

DOI: 10.1021/acs.chemmater.7b02174 Chem. Mater. 2017, 29, 7330−7344

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Chemistry of Materials were dissolved in toluene and stored in the dark until use. The concentrations of ZnSe QD stock solutions were estimated by TEM and ICP-OES measurements.48 ZnSe/ZnS Synthesis. Typical procedures for ZnS overcoating process are described here. ODE (8.0 mL), oleylamine (6.0 mL), TOP (4.0 mL), 1-dodecylphosphonic acid (17.4 mg, 7.0 × 10−5 mol), and ZnSe QD cores (1.2 × 10−7 mol in 0.5 mL of toluene) were loaded into a 100 mL four-neck round-bottom flask. The reaction mixture was degassed under vacuum at 100 °C to remove toluene and other volatiles and filled with N2. The amount of shell precursors required to form five monolayers of ZnS was calculated following literature procedures.53 Zn oleate solution (0.2 M) and n-octanethiol solution (0.2 M) were added dropwise separately via syringe pump starting at 280 °C, and the reaction mixture was heated to 310 °C during the overcoating process. A 1.5-fold excess of n-octanethiol was used compared to Zn oleate. After the precursor addition was done, the reaction mixture was further annealed at 200 °C for 60 min. CdxZn1−xSe/CdyZn1−yS/ZnS Synthesis. Typical procedures for cation exchange and subsequent overcoating process are described here. ODE (3.0 mL), oleylamine (5.0 mL), TOP (3.0 mL), 1dodecylphosphonic acid (20.2 mg, 8.1 × 10−5 mol), and ZnSe QD core (1.2 × 10−7 mol in 0.9 mL of toluene) were loaded into a 100 mL four-neck round-bottom flask. The reaction mixture was degassed under vacuum at 100 °C to remove toluene and other volatiles and filled with N2. The amount of shell precursors required to form four monolayers of Cd0.2Zn0.8S and three monolayers of ZnS was calculated following literature procedures.53 To form CdZnSe cores via cation exchange, the desired volume of 0.2 M Cd oleate solution was injected at 220 °C. After 15 min, a mixture of 0.2 M Cd oleate and 0.2 M Zn oleate solution and 0.2 M n-octanethiol solution were added dropwise separately via syringe pump. The reaction temperature was raised to 290 °C during precursor addition. A ∼1.2−1.5-fold excess of noctanethiol was used compared to the metal precursors. The addition rates of the metal and sulfur precursors were adjusted to be proportional to the molar ratios of the precursors used. After the CdZnS shell precursor addition was done, 0.2 M Zn oleate solution and 0.2 M n-octanethiol solution were added dropwise separately using a syringe pump as described above. The reaction temperature of ZnS shell formation was set between 290 and 310 °C. A ∼1.5-fold excess of n-octanethiol was used compared to the Zn precursors. The total reaction time of the overcoating process was ∼4−6 h depending on the desired shell thickness. For some of the QD synthesis, an additional amount of Zn oleate was further added dropwise to improve the Zn-rich outermost layer.54 After the precursor addition was done, the reaction mixture was annealed at 200 °C for 60 min. ZnSe/CdyZn1−yS/ZnS Synthesis. Typical procedures for the direct overcoating process are described here. ODE (3.0 mL), oleylamine (5.0 mL), TOP (3.0 mL), and ZnSe QD core (1.2 × 10−7 mol in 0.9 mL of toluene) were loaded into a 100 mL four-neck round-bottom flask. The reaction mixture was degassed under vacuum at 100 °C to remove toluene and other volatiles and filled with N2. The amount of shell precursors required to form four monolayers of Cd0.4Zn0.6S and two monolayers of ZnS was calculated following literature procedures.53 Zn oleate solution (0.2 M, 0.34 mL, 6.8 × 10−5 mol) was injected at 90 °C. The target reaction temperature was set to 290 °C by a temperature controller. Once the reaction temperature reached 220 °C, a mixture of 0.2 M Cd oleate and 0.2 M Zn oleate solution and 0.2 M n-octanethiol solution were added dropwise separately via syringe pump. The reaction temperature was raised to 290 °C during the precursor addition. A ∼1.2−1.5-fold excess of noctanethiol was used compared to the metal precursors. The addition rates of the metal and sulfur precursors were adjusted to be proportional to the molar ratios of the precursors used. After addition of the CdZnS shell precursors was done, 0.2 M Zn oleate solution and 0.2 M n-octanethiol solution were added dropwise separately via syringe pump as described above. The reaction temperature of ZnS shell formation was set between 290 and 310 °C. A ∼1.5-fold excess of n-octanethiol was used compared to the Zn precursors. The total reaction time of the overcoating process was ∼4−6 h depending on the desired shell thickness. For some of the QD synthesis, an

additional amount of Zn oleate was further added dropwise to improve the Zn-rich outermost layer.54 After the precursor addition was completed, the reaction mixture was annealed at 200 °C for 60 min. FITC Coupling and pH Sensing Assay. Typical reaction conditions used were as follows: 464 nm emitting QD coated with DHLA-PEG750-OMe:DHLA-PEG600-NH2 (4:1) (12.0 μM, 250 μL, 3.0 × 10−9 mol) was mixed in 500 μL of 0.1 M NaHCO3/Na2CO3 buffer (pH 9). FITC solution (3.41 mM in DMSO, 18 μL, 6.1 × 10−8 mol) was added to the QD solution to initiate the reaction. After gentle stirring for 7 h in the dark at room temperature, the mixture was directly loaded onto a PD-10 desalting column (GE Healthcare Life Sciences) and eluted with 0.1× PBS.55 Elution of the QD product (band) was traced by hand-held UV lamp. The first emitting single band was collected and transferred to a centrifugal purification unit (Amicon Ultra 30K, Millipore). The product solution was diluted with deionized water and centrifuged at 3800 rpm for ∼5−10 min, and the filtrate was discarded. The concentrated QD solution was used for subsequent pH sensing experiments. Buffer solutions used are as follows: 0.1 M AcOH + 0.1 M NaOAc for pH 3−6; 50 mM Tris + HCl for pH 6.5−9. Cell Culture and Cytotoxicity Test. African green monkey kidney cells (COS-1) were maintained in complete growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM; American Type Culture Collection, ATCC) supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS; ATCC) and 1% (v/v) antibiotic/ antimycotic solution (Sigma-Aldrich) at 37 °C and 5% CO2. The cells were cultured in T25 or T75 flasks under a humidified atmosphere and passaged at 80% confluency. The water-soluble QDs were conjugated to the membrane localization peptide JB85856 or the cell penetrating peptide (CPP) JB43456 by incubating the assemblies at a QD:peptide ratio of 1:30 (for JB858) or 1:20 (for JB434) in DMEM supplemented with 25 mM HEPES (DMEM/HEPES, pH 7.4; Life Technologies, Carlsbad, CA). The sequence of the membrane-targeting peptide JB858 used in this study is WGDabPalVKIKKP9GGH6, where Dab and Pal are the artificial residue diaminobutyric acid and palmitoyl group, respectively. The palmitoyl group is anchored to the Dab residue by a nonhydrolyzable amide linkage. The sequence of the CPP JB434 is R9GGLAAAibSGWKH6, where Aib is the artificial residue αaminoisobutyric acid. To ensure that no aggregation occurred in response to the incubation, each component was mixed following a specific protocol. The peptides were initially suspended in DMSO, and the DMSO solution was further mixed with DMEM/HEPES buffer so that the final DMSO concentration was 5% (v/v). The QD solutions were added to the peptide solution, vortexed, and allowed to incubate for 30 min, after which the solution was spun briefly to ensure that no aggregation had occurred. The final concentration of the QDs was 500 nM. For cytotoxicity analysis, cellular proliferation was analyzed using the CellTiter 96 cell proliferation assay (Promega, Madison, WI) by following the manufacturer’s prescribed instructions. The assay is based upon the conversion of a tetrazolium substrate to a formazan product by viable cells at the assay end point. Briefly, 96-well plates were seeded with ∼2 × 103 cells/well and allowed to incubate overnight. The cells were then exposed to increasing concentrations of the QDs for 30 min in DMEM/HEPES. The solutions were removed, washed with DMEM/HEPES, and replaced with complete media. The cells were cultured for a further 48 h before performing the assay. A control of DMEM delivery was performed to standardize the results, and media alone was used as a blank control. QD-FITC Delivery to Cellular Membranes Using Peptide JB858. For QD-FITC delivery and attachment to cellular membranes, 14 mm dishes (MatTek Corporation; Ashland, MA) were seeded with ∼2 × 105 cells per dish and allowed to incubate overnight. To increase adhesion, the plates were pretreated with fibronectin for at least an hour prior to plating. The QD-FITC conjugates were assembled with the membrane targeting peptide JB858 by incubating the conjugates at a QD:peptide ratio of 1:40 in DMEM/HEPES. The final concentration of the QDs and JB858 were 200 nM and 8 μM, respectively. The QD-FITC-JB858 assemblies were incubated on 7333

DOI: 10.1021/acs.chemmater.7b02174 Chem. Mater. 2017, 29, 7330−7344

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Figure 1. Absorption (A) and fluorescence (B) spectra of ZnSe core QDs (purple line), the QDs after cation exchange (red line), the QDs after overcoating with Cd0.2Zn0.8S layers (green line), and the QDs after overcoating with ZnS layers (blue line). The QD samples are dissolved in chloroform. (C and D) High-resolution TEM images of ZnSe QDs before and after cation exchange: (C) the as-prepared ZnSe QDs (average size 4.8 ± 0.30 nm; the lowest absorption at 410 nm) and (D) the product after cation exchange with Cd oleate (average size 5.0 ± 0.29 nm; the lowest absorption at 449 nm). prewashed cells for 30 min, washed, and the media was changed to Dulbecco’s phosphate-buffered saline (DPBS).56 Acquisition and Image Analysis of Extracellular pH Sensing. The cells were subsequently imaged on a Nikon A1RSi confocal microscope using spectral acquisition from 440 to 625 nm binned over 5 nm. A 405 nm laser was used to excite the sample in the same manner as normal confocal microscopy, and the emission beam from the sample was then polarized and sent through a diffraction grating cassette which was set at 5 nm resolution. The output was then focused onto a 32 photomultiplier tube (PMT) array which acquires the output from each of the 5 nm bins and produces images and intensity data. The resulting image is a compilation of each of the wavelengths and provides intensity data for each specific wavelength bin. To explore pH effects on the fluorescent profiles of the QD-FITC conjugates attached to the cell membranes, the cells were iteratively incubated in buffers of decreasing pH values. In between each media exchange, the cell monolayers were washed twice. After each image was taken, multiple regions of interest (ROIs) were chosen at centers on the membrane, and the spectral intensity data were exported into Excel for analysis. Images were processed using Nikon Elements 4.50.00. As a control, the same QD-FITC conjugates were assembled with JB858 at a QD:peptide ratio of 1:15 in DPBS with each desired pH, and the fluorescence spectra of each sample were measured using the Tecan microtiter plate reader. Detailed descriptions of ligand exchange and characterization procedures, including pH and salt stability tests, Förster resonance energy transfer (FRET) analysis, and agarose gel electrophoresis, can be found in the Supporting Information.

monodispersed spherical shapes (Figure 1 and Figure S1). After washing away excess ligands, the as-prepared ZnSe QD cores were subsequently used for cation exchange with Cd2+ ions to form CdxZn1−xSe cores, and this was further followed by the overcoating process. Cd oleate was used as the Cd2+ source, and the amount of Cd oleate was adjusted to tune the fluorescence wavelength. Addition of Cd oleate in the ZnSe core solution at 220 °C drove a progressive red-shift of the lowest absorption band. While the sharp lowest absorption band was relatively well-preserved during the cation exchange process, slight broadening was observed for the fluorescence band (Figures 1 and S2). The TEM images of the QDs before and after cation exchange showed no apparent change of the size and shape (Figure 1), indicating that the original ZnSe core structure was well-preserved during the reaction. This suggests that the polydispersity of the CdxZn1−xSe QDs should not be the cause of the observed fluorescence broadening. Rather, one possible reason for this could be inhomogeneous distribution of Cd2+ ions in the CdxZn1−xSe cores under the present experimental conditions. Cd x Zn 1−x Se QDs formed via cation exchange were subsequently overcoated with CdyZn1−yS layers to protect the emissive cores and further tune the fluorescence wavelength. Overcoating with a CdyZn1−yS shell provides some inherent benefits compared to just direct overcoating with a ZnS shell because inclusion of Cd helps minimize the lattice mismatch with CdxZn1−xSe core and a change in Cd molar ratio of CdyZn1−yS precursors can also contribute to tuning of the fluorescence wavelength. ZnSe/CdS core/shell QDs are known to have type II features,7,57,58 and this is usually characterized by the disappearance of the lowest absorption band concomitant with a significant red-shift and broadening of the fluorescence as the band gap is lower than those of ZnSe and CdS. The present core/shell structural design is analogous to ZnSe/CdS in the case where x is close to zero and y is large. To

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RESULTS AND DISCUSSIONS Synthesis of CdxZn1−xSe/CdyZn1−yS/ZnS and ZnSe/ CdyZn1−yS/ZnS Core/Multishell Alloyed Quantum Dots. Initial ZnSe QD cores were synthesized from ZnEt2 and TOP:Se via a conventional hot injection method.13,27,51,52 During core growth, additional precursors were added dropwise until the lowest absorption band reached the desired wavelength. ZnSe QD cores prepared in this manner exhibit narrow lowest absorption and fluorescence bands along with 7334

DOI: 10.1021/acs.chemmater.7b02174 Chem. Mater. 2017, 29, 7330−7344

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Figure 2. (A) Change of QYs (red circle) and fwhms of the QD fluorescence bands (cyan square) during cation exchange and the subsequent overcoating processes of ZnSe cores. The purple, green, and blue arrows indicate the process of cation exchange, Cd0.2Zn0.8S overcoating, and ZnS overcoating, respectively. (B) Evolution of the fluorescence peak shift during CdyZn1−yS and ZnS layer overcoating of ZnSe cores with different Cd molar ratios (y). The arrows indicate the starting point of ZnS overcoating process following CdyZn1−yS overcoating.

Table 1. Selected Properties of Representative Core/Shell QDs Prepared in this Study QD composition

CdyZn1−yS (ML)

ZnS overcoating ZnSe/ZnS CdZnSe/Cd0.2Zn0.8S/ZnS CdZnSe/Cd0.1Zn0.9S/ZnS CdZnSe/Cd0.2Zn0.8S/ZnS CdZnSe/Cd0.2Zn0.8S/ZnS CdZnSe/Cd0.3Zn0.7S/ZnS CdZnSe/Cd0.3Zn0.7S/ZnS

4 4 4 4 3 3

ZnSe/Cd0.2Zn0.8S/ZnS ZnSe/Cd0.4Zn0.6S/ZnS ZnSe/Cd0.3Zn0.7S/ZnS ZnSe/Cd0.35Zn0.65S/ZnS ZnSe/Cd0.4Zn0.6S/ZnS ZnSe/Cd0.5Zn0.5S/ZnS ZnSe/Cd0.4Zn0.6S/ZnS

3 3 3 4 4 3 4

ZnS (ML)

TEM size (nm)

λem (nm)

fwhm (meV) [in nm]

4.5 7.9 ± 0.8 420 cation exchange → CdyZn1−yS overcoating → ZnS overcoating 2 8.6 ± 0.9 475 2 7.5 ± 0.6 483 3 7.7 ± 0.7 489 2 7.4 ± 0.6 502 3 7.6 ± 0.5 527 3 8.2 ± 0.7 527 CdyZn1−yS overcoating → ZnS overcoating 3 8.3 ± 0.9 448 3 8.8 ± 0.7 458 3 7.1 ± 0.5 464 3 7.7 ± 0.6 479 2 8.6 ± 0.7 479 3 8.2 ± 0.8 509 3 8.4 ± 0.8 513

QY

113 [16.1 nm]

0.26a

138 196 159 163 164 165

[25.0 [37.0 [30.5 [33.2 [36.4 [36.8

nm] nm] nm] nm] nm] nm]

0.38a/0.28b (74%)c 0.38a/0.27b (71%)c 0.34a/0.26b (76%)c 0.41a 0.41a 0.53a

144 127 145 135 131 166 177

[23.4 [21.6 [25.4 [25.1 [24.1 [34.6 [37.7

nm] nm] nm] nm] nm] nm] nm]

0.32a 0.35a/0.29b (83%)c 0.32a/0.24b (75%)c 0.35a/0.20b (57%)c 0.40a 0.41a 0.49a

a

QY of as-prepared QD measured in toluene. bQY measured in water after ligand exchange. cPercentage QY retained after ligand exchange and transfer to water.

keep the narrow fluorescence band in the blue region, it is ideal to keep the type I feature of the core/shell QDs; hence, we did not increase the Cd molar ratio in CdyZn1−yS layers beyond 50% (i.e., y ≤ 0.5). Overcoating with CdyZn1−yS layers was achieved by slowly adding a mixture of Cd oleate and Zn oleate as the metal precursors to the reaction along with n-octanethiol as the sulfur precursor at ∼290−310 °C. Use of n-octanethiol at high temperature has been effective in forming CdS, ZnS, or CdZnS layers with excellent size uniformity, and the importance of a slow decomposition rate of n-octanethiol has been addressed.59−61 Small aliquots of the reaction mixture were periodically taken, and the absorption and fluorescence spectra were monitored throughout the overcoating process (Figure 1). Both the absorption and fluorescence peaks showed a progressive red shift in the early stage of CdyZn1−yS overcoating step, and this gradually slowed down in the later stages. These red shifts suggest a delocalization of the exciton over the CdyZn1−yS shell and/or further infusion of Cd2+ into the CdxZn1−xSe core. QYs of the QDs gradually increased concomitant with narrowing of the full width at half-maximum (fwhm) of the fluorescence band during the CdyZn1−yS overcoating (Figure 2A). The QY of the CdxZn1−xSe core after cation exchange was low (∼1−2%) presumably because surface defects were created during the cation exchange. The

overcoating of the CdxZn1−xSe cores with CdyZn1−yS layers significantly improved the QYs up to ∼60%. QYs after overcoating with an additional two monolayers of ZnS did not show any significant increases over those measured after CdyZn1−yS coating. QYs of a series of the as-prepared QDs after overcoating were typically in the ∼30−55% range in this study, see Table 1. Such enhancement of QYs has been commonly observed during overcoating of QD cores with higher band gap materials due to confinement of excitons in the core and passivation of the surface trap states.62 The fwhm of the fluorescence band was also monitored during the overcoating processes because it can help assess underlying size dispersity in the QD samples. The initial cation exchange of ZnSe QD cores with Cd2+ ions usually led to broadening of the fluorescence band. However, the fluorescence band gradually narrowed during overcoating with CdyZn1−yS layers. The latter is presumably due to the homogeneous distribution of Cd2+ ions in the CdxZn1−xSe cores over time at the high reaction temperature used in this study (∼300 °C), which is expected to promote the alloying process. In contrast, subsequent overcoating with ∼2−3 monolayers of ZnS caused a sizable blue shift (typically ∼5− 10 nm) of both the absorption and fluorescence bands (Figure 1), suggesting an interfacial alloying process between 7335

DOI: 10.1021/acs.chemmater.7b02174 Chem. Mater. 2017, 29, 7330−7344

Article

Chemistry of Materials

Figure 3. High-resolution TEM images of the as-prepared core/multishell QDs: (A) CdZnSe/Cd0.3Zn0.7S(3 ML)/ZnS(3 ML) QDs (average size: 8.2 ± 0.7 nm, λem = 527 nm); (B) ZnSe/Cd0.3Zn0.7S(3 ML)/ZnS(2 ML) QDs (average size: 9.0 ± 0.6 nm, λem = 459 nm); (C) ZnSe/Cd0.4Zn0.6S(3 ML)/ZnS(3 ML) QDs (average size: 8.8 ± 0.7 nm, λem = 458 nm). ML: monolayer.

ratios (y = 0.5) were shifted beyond 500 nm; here, the energy level is lower than ZnSe bulk band gap energy (∼2.7 eV). This implies that the infusion of Cd2+ ions into ZnSe cores to form CdxZn1−xSe, along with exciton delocalization to CdyZn1−yS shells, may be the major reason for the wide range of spectral tuning. Consistent with the narrow fluorescence bands, TEM images of the as-prepared multishell alloyed QDs displayed quasispherical shapes with narrow size distributions and shape uniformity (Figure 3, Figure S6, and Table 1). The TEM sizes of the QDs matched relatively well with those estimated from the ZnSe core size and amount of the shell precursors used for overcoating, suggesting that the core/multishell QD structures with designed compositions were forming as expected. High resolution TEM images of the QDs also revealed high crystallinity along with continuous lattice fringes throughout the entire particle (Figure 3 and Figure S6). Some of the QD samples formed uniform two-dimensional arrays over large areas, suggesting high nanocrystalline size monodispersity (Figure 3C). Powder X-ray diffraction (XRD) patterns of the original ZnSe core, CdxZn1−xSe prepared by cation exchange, and the overcoated sample along with the control zinc blende CdSe core are presented in Figure S7. XRD patterns of the asprepared ZnSe QDs match the theoretical peaks of the zinc blende crystal structure where the broad peaks are due to finite sizes of the QD samples.1,63 The XRD patterns of ZnSe QD samples after cation exchange with Cd2+ ions shifted to smaller 2Θ values and appeared in between those of ZnSe and CdSe zinc blende QDs, suggesting the formation of CdxZn1−xSe alloy QDs. After overcoating with four layers of Cd0.2Zn0.8S followed by two layers of ZnS, the XRD patterns are shifted back to larger 2Θ values, reflecting the contributions of the ZnS layers on the diffraction pattern of the core/multishell QDs. Energy-dispersive X-ray (EDX) spectra were also acquired to explore the chemical compositions and provide some relative mapping of each element in the core/multishell structure (Figures S8 and S9). While Zn and Se related signals were

CdyZn1−yS and ZnS layers. During ZnS coating, the fwhm of the fluorescence band did not show any significant change in most cases. The capability to tune the fluorescence peaks with CdyZn1−yS overcoating allowed us to directly overcoat ZnSe QD cores without the initial fluorescence peak tuning via cation exchange and still achieve similar spectral features (Scheme 1). Similar to Figure 1, direct overcoating of the ZnSe core with CdyZn1−yS layers led to red shifting of the lowest absorption and fluorescence bands, and the subsequent ZnS overcoating then caused the blue shifts (Figure S3). Figure 2B shows the fluorescence peak shifts observed during CdyZn1−yS and ZnS layer overcoating of the ZnSe cores using different Cd molar ratios. As the Cd molar ratio was increased in the CdyZn1−yS overcoating step, the QD fluorescence peaks exhibited further red shifting, suggesting that the Cd ions are infused into the ZnSe cores and a CdxZn1−xSe alloy with a higher Cd ratio is formed. In contrast, direct overcoating of ZnSe cores with ZnS layers showed only a few nm red shift (