Determination of Concentration of Amphiphilic Polymer Molecules on

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Determination of Concentration of Amphiphilic Polymer Molecules on the Surface of Encapsulated Semiconductor Nanocrystals Aleksandra Fedosyuk,§ Aliaksandra Radchanka,§ Artsiom Antanovich,§ Anatol Prudnikau,§ Maksim V. Kvach,† Vadim Shmanai,† and Mikhail Artemyev*,§ §

Institute for Physical Chemical Problems, Belarusian State University, Leningradskaya str. 14, Minsk 220030, Belarus Institute of Physical Organic Chemistry, National Academy of Sciences of Belarus, Minsk 220072, Belarus

†

S Supporting Information *

ABSTRACT: We present a method for the determination of the average number of polymer molecules on the surface of AIIBVI luminescent core−shell nanocrystals (CdSe/ZnS, ZnSe/ZnS quantum dots, and CdS/ZnS nanorods) encapsulated with amphiphilic polymer. Poly(maleic anhydride-alt-1-tetradecene) (PMAT) was quantitatively labeled with amino-derivative of fluorescein and the average amount of PMAT molecules per single nanocrystal was determined using optical absorption of the dye in the visible spectral range. The average amount of PMAT molecules grows linearly with the surface area of all studied nanocrystals. However, the surface density of the monomer units increases nonlinearly with the surface area, because of the increased competition between PMAT molecules for Zn-hexanethiol surface binding sites. The average value of zeta potential (ζ = −35 mV) was found to be independent of the size, shape, and chemical composition of nanocrystals at fixed buffer parameters (carbonate−bicarbonate buffer, pH 9.5 and 5 mM ionic strength). This finding is expected to be useful for the determination of the surface density of remaining carboxyl groups in PMAT-encapsulated nanocrystals.

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INTRODUCTION Highly luminescent semiconductor colloidal nanocrystals (NCs), such as CdSe, InP, and PbS, that have found application in biological assays and detection platforms,1−3 are primarily prepared using high-temperature solution routes and are essentially capped with hydrophobic ligands such as TOP/ TOPO, amines, and long-chain carboxylates.4−6 Since biolabeling applications require water-soluble NCs, additional postsynthetic treatment is necessary to make these NCs compatible with biological media. Several strategies of achieving this goal were developed, and the majority of them can essentially be divided into two main categories: encapsulation and ligand exchange.7−9 Encapsulation with an amphiphilic polymer is used to achieve colloidal stability of NCs in aqueous solutions and provide a source of functional groups that can be further used for chemical functionalization of the surface of the nanoparticles (NPs).7,8,10 A polymer coating also allows one to control the hydrodynamic size and surface charge of NPs.8,11−13 Surface charge of NPs is a key characteristic that determines colloidal stability,14,15 as well as the structure and function of nanocomposites; it affects the self-assembly of nanoparticles and the preparation of nanoarchitectures,16−18 and it influences the performance of NPs in a variety of biomedical assays.19,20 Molecular packing density and the corresponding molecular conformation of functional ligands on the surface of NPs are expected to be important parameters for regulatory requirements and quality control in clinical applications.21,22 To © 2016 American Chemical Society

control surface properties of NPs for subsequent functionalization, it is necessary to have information about the number and charge of the functional groups on the surface of the NPs, including those intended for conjugation with biomolecules. In the case of NCs encapsulated into amphiphilic polymer, the number and exact arrangement of polymer molecules on their surface, as well as the coverage density, strongly influence the colloidal and chemical stability of NCs in a biological environment, as well as their hydrodynamic size and zeta potential (ζ). Here, we quantify the average amount of amphiphilic polymer molecules on the surface of colloidal semiconductor nanocrystals made of different materials, including CdSe, CdS, and ZnSe, which have different size and shape (spherical (quantum dots) and elongated (nanorods)). All NCs are coated with a ZnS shell in order to maintain similar surface chemistry. To quantify polymer chains, we used dye-labeled poly(maleic anhydride-alt-1-tetradecene) with a controlled amount of dye molecules per polymer chain. Using the DLS technique, we established the correlation of the surface area of NCs with the average number of polymer chains and zeta potential at fixed pH. Although dye labeling of the amphiphilic polymeric shell was earlier utilized to study Förster resonance Received: December 16, 2015 Revised: February 4, 2016 Published: February 11, 2016 1955

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Langmuir energy transfer (FRET) between dye and QDs,23,24 to the best of our knowledge, quantitative determination of the number of amphiphilic polymer molecules on the surface of encapsulated semiconductor NCs has not been performed yet.

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fold excess to NRs. To obtain approximately equal magnitudes of the absorption peaks of the dye and NCs, the ratio NPMAT/NPMAT‑Dye = k1 was varied, considering the molar extinction coefficient of each type of NC. The resultant solid phase of encapsulated NCs, along with the excess of polymers, was dissolved in aqueous NaOH solution. Removal of excessive polymer was carried out via gel filtration of an aqueous solution of encapsulated NCs, using a column filled with Sephacryl S500. Aqueous colloidal solutions of encapsulated NPs were passed through the column in carbonate−bicarbonate buffer at pH 9.5. Retention times of NPs and free polymer−dye in exiting fractions were determined using photoluminescence (PL) and optical absorption spectra. The first fraction (ca. 30%, by volume) containing only encapsulated NCs (retention time between 10 and 15 min for 50 mm gel) showed both absorption and PL bands of NCs and the dye, while spectra of the last fraction with the retention time between 15−20 min showed only absorption and PL bands of the dye. The retention time for the free dye-doped PMAT reference sample was >15 min under the same experimental conditions. The absence of the free dye-doped polymer in a colloidal solution of nanocrystals with a retention time of 10−15 min was additionally checked via gel electrophoresis (see Figure S2 in the Supporting Information). For all further experiments, only the first fraction with a retention time of 10−15 min was used. Determination of the Concentration and Zeta Potential of NCs Encapsulated with Dye-Labeled PMAT. The concentration of CdSe/ZnS, CdS/ZnS, and ZnSe/ZnS QDs encapsulated with PMAT dye was determined using the molar extinction coefficients of corresponding core NCs. For CdSe and CdS QDs, we used published data,32 whereas, in the case of ZnSe QDs and CdS NRs, molar extinction coefficients were determined analytically, using inductively coupled plasma−atomic emission spectroscopy (ICP-AES). The general procedure for the ICP-AES analysis was carried out analogously to that reported in ref 33. Briefly, an aliquot of CdS NRs in chloroform with a known UV-vis optical absorption spectrum was mixed with methanol and centrifuged at 4000 rpm for 10 min. The solvent then was discarded and the solid precipitate was dried at room temperature overnight. Dry NCs were dissolved in concentrated nitric acid at 90 °C for 24 h, and the resultant solution of cadmium nitrate was diluted with doubly distilled water. The concentration of Cd ions in the resultant solution was determined using ICP-AES analyzer (Varian Liberty Sequential) with a reference to the cadmium nitrate analytical standard solution. Similar procedure was performed for ZnSe core NCs of various diameters. The plot of the resultant molar absorption coefficient at first exciton versus the diameter of the ZnSe core is presented in Figure S3 in the Supporting Information. Note that the deposition of a ZnS shell 1−2 monolayers (MLs) thick atop CdSe, CdS, or ZnSe core NCs introduces insignificant changes in the optical absorption of core NCs at the first excitonic peak.34 Zeta potential of PMAT-encapsulated colloidal NCs was measured in a carbonate−bicarbonate buffer solution at pH 9.5 and 5 mM ionic strength using Malvern Zetasizer Nano ZS90 instrument.

METHODS

Chemicals. Poly(maleic anhydride-alt-1-tetradecene) (PMAT, molecular weight (Mw) = 9000, formula weight (FW) = 294.4), N(3-(dimethylamino)propyl)-N′-ethylcarbodiimide (EDC), NaOH, NFmoc-1,4-butanediamine hydrobromide, 1-hexanethiol, cellulose acetate membrane (cutoff = 12 kDa), Sephacryl S-500 (GE Healthcare, Inc.) were purchased from Sigma−Aldrich. 4-(Fluorescein-6-carboxamido)-butylammonium chloride was prepared from pentafluorophenyl ester derivative of cyclohexanecarbonyl-protected 6-carboxyfluorescein,25 according to the synthetic procedure described in the Supporting Information. PMAT Dye Labeling. 45 mg of PMAT and ca. 7 mg of 4(fluorescein-6-carboxamido)-butylammonium chloride were dissolved in 5 mL of dry methanol at 50 °C. Then, N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide (EDC) was added in a 10-fold excess to the dye and the reaction mixture was stirred at 50 °C for 2 h. The solution then was cooled and stirred overnight at room temperature in order to complete the reaction between PMAT carboxyl groups and dye amino groups. Then, methanol was evaporated and the solid phase was dissolved in aqueous NaOH solution. Dye-labeled polymer was purified from the excess of free dye by dialysis against distilled water using cellulose acetate membrane. After dialysis, an aliquot of the purified sample was dried, weighed, and redissolved in a buffer solution with pH 9.5. The solution was transferred into a quartz cuvette, and an ultraviolet−visible light (UV-vis) absorption spectrum was recorded with an Ocean Optics Model HR2000+ spectrophotometer. The average number of the dye molecules per single PMAT chain was calculated from the weighted amount of the polymer in the dried aliquot and the concentration of the dye that was determined from the absorption spectrum of PMAT dye in the buffer, assuming that the molar absorption coefficient for the dye is identical to that of fluorescein (molar absorption of fluorescein in basic aqueous solutions: ε ≈ 80 000 cm−1 M−1). The rest of the dialyzed aqueous solution of PMAT dye was dried at 60 °C and then dry polymer was redissolved in chloroform. The resultant solution was filtered through a 0.2 μm nylon membrane and stored at +4 °C in darkness. Synthesis and Characterization of NCs. CdSe/ZnS core−shell QDs of different sizes (4−7 nm) were synthesized according to ref 26. CdS/ZnS core−shell QDs were prepared as described in refs 27 and 28. CdS/ZnS core−shell NRs, 4.0 nm × 22.0 nm in size, were synthesized as described in refs 29 and 30. ZnSe/ZnS core−shell QDs with a diameter of 6.7 nm were synthesized according to the published procedure.31 All NCs were purified by precipitation with methanol, centrifugation at 5000 rpm for 10 min, and finally redissolution in fresh chloroform. Average surface areas were calculated from sizes of corresponding core−shell NCs that were determined using transmission electron microscopy (TEM). TEM samples were prepared by drying a droplet of NCs chloroform solution on carbon-coated copper grids. TEM micrographs were acquired using a LEO Model 906E TEM system. Encapsulation and Solubilization of NCs with Dye-Labeled PMAT. At first, NCs were treated with hexanethiol to obtain similar surface coverage for all studied NCs. Briefly, 10 μL of hexanethiol were added to 1 mL of the solution of NCs in chloroform that contained ca. 5 mg of NCs and a mixture was stirred for 1 h at room temperature. The hexanethiol-capped NCs then were precipitated with methanol, centrifuged at 5000 rpm (2500 g) for 10 min, washed several times with methanol, and redispersed in a fresh portion of chloroform. To this solution, a mixture of pure PMAT and dye-labeled PMAT in chloroform at different PMAT/dye-to-PMAT proportion was added. The obtained mixture was stirred for 24 h and dried at room temperature in darkness in order to prevent possible photodegradation of the dye. To achieve complete encapsulation, the total amount of added polymers was in a 2.5-fold weight excess to all QDs and in a 6-

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RESULTS AND DISCUSSION The general idea of this experiment was to use an amphiphilic polymer statistically labeled with the controlled amount of dye with a known molar extinction coefficient as a marker for estimating the number of polymer chains on the surface of encapsulated NCs. The amount of polymer chains per single NC was determined using optical methods, i.e., by comparing optical absorption of the dye and NCs at the first excitonic band. General reaction scheme of PMAT labeling with the dye is depicted in Figure 1. Standard carbodimide-mediated reaction between aminogroups of 4-(fluorescein-6-carboxamido)-butylammonium chloride and carboxyl or anhydride groups of PMAT results in the statistical binding of the dye molecules to the PMAT chain. The average amount of the dye molecules per single PMAT chain was estimated using optical spectroscopy. To do 1956

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Figure 2. UV-vis absorption spectra of CdSe/ZnS QDs encapsulated with pure PMAT (black) and PMAT−dye (red). The spectrum of the neat dye (blue) was obtained by the subtraction of QDs/PMAT spectrum from QDs/PMAT−dye one. All measurements were conducted in a carbonate−bicarbonate buffer (pH 9.5 and 5 mM ionic strength).

Figure 2 shows that both QDs and PMAT−dye have wellresolved absorption bands that allow one to obtain the absorption spectrum of PMAT dye separately from the QDs contribution. For that, the absorption spectrum of the same QDs encapsulated with pure PMAT (black curve in Figure 2) was normalized to the absorption spectrum of QDs/PMAT− dye (red curve) at the maximum of the first excitonic transition of QDs (λ = 545 nm). After that, the QDs/PMAT spectrum was subtracted from the QDs/PMAT−dye spectrum. The resultant spectrum (blue curve in Figure 2) is the absorption spectrum of PMAT-dye. Figure S4 in the Supporting Information represents optical absorption spectra of PMATdye before encapsulation in pH 9.5 buffer solution. Now, the average amount of PMAT molecules per single NC can be calculated using eq 1:

Figure 1. Reaction scheme for PMAT labeling with 4-(fluorescein-6carboxamido)-butylammonium chloride and schematic structure of the encapsulated nanocrystal (NC).

this, the aliquot of the dye-labeled polymer containing a known amount of PMAT was transferred into the carbonate− bicarbonate buffer with pH 9.5 and the UV-vis absorption spectrum was registered. By using known molar extinction coefficients and taking into account the average molecular weight of PMAT (Mw = 9000), we determined the molar concentration of the dye in the aliquot and the average ratio of the dye molecules per single PMAT chain NDye/NPMAT = k2 = 1.53. The coefficient k2 = 1.53 was used to determine the average number of PMAT chains per single encapsulated NC of different size and geometry. For that, colloidal solutions of encapsulated NCs after the separation of unbound PMAT (see the Methods section) were transferred into the carbonate− bicarbonate buffer with pH 9.5 and the UV-vis absorption spectra were recorded. Naturally, correct determination of the relative dye/NC absorption can be achieved when intensities of both absorption bands are nearly equal. After comparison of the fluorescein molar extinction coefficient (ε ≈ 80 000 cm−1 M−1 under basic conditions) and the molar extinction coefficient for CdSe QDs with a diameter of ∼4 nm (∼2.3 × 105 cm−1 M−1, according to ref 32), we came to the conclusion that the correct measurements require roughly 3 dye molecules per single NC. Preliminary experiments showed that encapsulation with pure dye-modified polymer yields NCs carrying ∼23 dye molecules per particle. In such a case, a strong contribution of the dye extinction obstructs correct determination of the concentration of the NCs. Thus, in our experiments, we used mixtures of dyemodified and nonmodified PMAT in different proportions. Figure 2 shows representative absorption spectrum of PMAT-dye-encapsulated CdSe/ZnS QDs with the first excitonic absorption peak at 545 nm and a diameter of 4.7 nm.

NPMAT D ε (k + 1) = 1 2 1 NNC ε1D2k 2

(1)

where NPMAT/NNC is the average number of PMAT chains per single nanocrystal, D1 and D2 are the optical densities of the dye and NCs at their absorption peaks, ε1 and ε2 are the molar absorption coefficients of the dye and NCs respectively, k1 is the experimentally variable coefficient defining the molar ratio of the dye-modified and nonmodified polymer, k2 is the average number of the dye molecules per single PMAT chain. Similar procedure was applied further to all studied NCs. For the experiment, seven types of QDs (spherical and quasispherical NCs) with different core composition (CdSe, CdS, and ZnSe) and diameter and one type of NR with a CdS core were selected. Table 1 summarizes the characteristics of all types of studied NCs. Representative TEM images with corresponding size histograms are presented in Figure S5 in the Supporting Information. Although the NC cores are composed of different materials, all of them are covered with the same ZnS shell, which allows one to maintain identical surface chemistry for all types of studied NCs. In addition to the fact that all types of core−shell NCs underwent the same surface treatment with hexanethiol to ensure the identical character of the van der Waals interaction of PMAT with surface ligands during the 1957

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Langmuir Table 1. Characteristics of Nanocrystals (NCs) Used in Experiments abbreviation

core

shell

core crystalline phase

QDs#1 QDs#2 QDs#3 QDs#4 QDs#5 QDs#6 QDs#7 NRs#1

CdSe CdSe CdSe CdSe CdS CdS ZnSe CdS

ZnS ZnS ZnS ZnS ZnS ZnS ZnS ZnS

wurtzite wurtzite wurtzite wurtzite zincblende wurtzite zincblende wurtzite

average diameter (nm)

average length (nm)

4.1 4.7 6.0 6.6 4.8 5.7 6.7 4.0

± ± ± ± ± ± ± ±

22.0 ± 0.5

0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.1

encapsulation. However, some variations of the chemical properties between different types of NCs could not be completely ruled out, because of the different crystalline structure of the core (zincblende ZnSe QDs versus wurtzite CdSe) that may affect the structure of the ZnS shell. Also note that CdS NRs were chosen over the more-popular CdSe, since CdS NRs have less defect surface, as compared to CdSe ones,35 which, in turn, may affect the surface roughness and defectiveness of the ZnS shell covering. Figure 3 shows the experimentally determined average number of PMAT molecules per single NC versus the diameter (Figure 3a) for QDs only and versus the surface area (Figure 3b) for both QDs and NRs. Figure 3b shows that, despite the different crystalline structure, shape, and size of core NCs, the average number of PMAT molecules per single NC follows the linear dependence y = −30.43 + 0.51x versus the surface area x of core−shell NCs above 4 nm in size well. Below 4 nm, this dependence deviates from linearity upon approaching zero value, probably due to the increased competition between polymer molecules for binding sites on the surface of the NCs. Figure 4 shows the area occupied by a single alkyl branch (MAT monomer) unit versus the NC surface area derived from the equation y = −30.43 + 0.51x (solid curve). Indeed, in Figure 4, we see that, on smaller NCs, PMAT molecules are arranged rather freely and the surface area occupied by single alkyl branch is >0.2 nm2. At the same time, in the case of the largest studied NCs, the surface concentration of MAT units approaches a value of ∼0.07 nm2 per single alkyl branch or MAT unit. Based on these data, we tried to model the surface arrangement of each PMAT unit on the surface of the ZnS shell. Since the exact crystalline structure of the surface of ZnS shell is unknown, it was simulated by the surface structure of bulk zincblende (ZB) and wurtzite (WZ) ZnS, represented by {100}, {110}, {111}, and {001̅} crystalline planes. Table 2 shows the calculated area per single Zn atom on different ZnS surfaces. If one assumes that the surface of spherical ZnS shell is equally represented by {100}, {110}, {111}, and {001̅} crystalline planes, then the average area per single Zn atom is equal to 0.178 nm2 for the ZnS wurtzite (WZ) phase and 0.160 nm2 for the ZnS zincblende (ZB) phases. These values are approximately twice as large as the value of 0.07 nm2 per single alkyl branch (MAT unit) observed on the largest studied NCs (see Figure 4). However, the value of surface area per single MAT unit obtained for intermediate-sized NPs lies within ∼0.15−0.35 nm2 range, which results in ca. 1:1 ratio of single alkyl branch per single thiol group (Zn atom). With the help of the scheme in Figure 5, we suggest the following PMAT

Figure 3. Average number of PMAT molecules per single NC versus (a) the QDs diameter and (b) the surface area of QDs and NRs. Black dots represent CdSe/ZnS QDs#1−4 from Table 1, red dots represent CdS/ZnS QDs#5 and QDs#6, blue dot represents ZnSe/ZnS QDs#7, and green diamond represents CdS/ZnS NRs#1. The solid curve in panel (b) is a linear fitting to the equation y = −30.43 + 0.51x. Error bars represent 25% standard deviation, as determined from the statistical analysis of three different measurements.

Figure 4. Average area per single alkyl branch of PMAT (single MAT unit) versus the average surface area of NCs. Black dots represent CdSe/ZnS QDs#1−4 from Table 1, red dots represent CdS/ZnS QDs#5 and QDs#6, blue dot represents ZnSe/ZnS QDs#7, and green diamond represents CdS/ZnS NRs#1. Solid curve is derived from the linear approximation y = −30.43 + 0.51x from Figure 3b, and dots represent the experimentally determined values from Figure 3b.

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strong competition of different PMAT molecules for binding sites that makes parts of the PMAT molecule remain unbound and form loops on the surface of PMAT-encapsulated NCs (brown chains in Figure 5). On the other hand, the deficient surface concentration of PMAT molecules on the small-area NCs can be explained by the existence of thiol-free areas on ZnS surface (S-terminated crystalline planes) to which PMAT cannot bind via vdW interaction. Such areas arise from the crystalline nature of AIIBVI core NCs and epitaxial character of ZnS shell. Unlike the shape of larger NCs, the shape of small AIIBVI cores cannot be treated as a simple sphere. It always contains facets of different crystalline planes. For example, both {100} and {111} crystalline planes of ZB may consist of either Zn or S atoms only. Since thiols bind only to the surface Zn atoms, sulfur-terminated facets are free from thiols and are not covered with PMAT. Therefore, the surface concentration of MAT units averaged over the entire surface area will be sufficiently reduced in the case of small NCs in accordance with the data provided on Figure 4 Finally, we examined the influence of PMAT surface concentration on the zeta potential (ζ) of encapsulated NCs. Figure 6 demonstrates the zeta potential versus the surface area and the number of carboxyl groups (N = 2nMAT units). Figure 6 shows that, for all types of studied NCs, the zeta potential value remains almost constant within the experimental error (ζ ≈ −35 mV) at fixed pH and ionic strength. For a given NP size, the ζ value of sub-100 nm NPs at moderate to low ionic strength of solution (Hü ckel regime) is linearly proportional to the surface charge density.36 The size dependence of ζ and fixed surface charge density (σ) then can be expressed as

Table 2. Calculated Area Per Single Zinc Atom on {100}, {110}, {111}, and {001}̅ Wurtzite and Zincblende ZnS Surfaces a (nm) 100 110 001̅ average

100 110 111 average

c (nm)

ZnS wurtzite 0.38226 0.62605 0.38226 0.62605 0.38226 0.62605

ZnS zincblende 0.54102 0.54102 0.54102

SZn (nm2) 0.239 0.169 0.127 0.178

0.146 0.207 0.127 0.160

arrangement on the surface of NCs of different size/surface area.

εε0kBTζ =

σzea 1 + ka

(2)

where kB is the Boltzmann constant, ε the dielectric permittivity of solvent, ε0 the permittivity of a vacuum, z the electrolyte valence, k the Debye parameter, and a the size of the NP. In the case when ka ≪ 1 (pure Hückel regime), the zeta potential ζ is dependent on the particle size, whereas, at the transition regime of ka > 1, ζ becomes size-independent. Figure 4 shows that the surface charge density σ increases nonlinearly with NP size and approaches an almost-constant value for the largest studied NPs. The almost-size-independent (within the experimental error) character of ζ in Figure 6 may also be explained by the increased screening effect of the neighboring carboxyl groups. Since the surface charge density is proportional to the number of charged groups per unit area, the average value of ζ = −35 mV at fixed buffer parameters (carbonate−bicarbonate buffer, pH 9.5 and 5 mM ionic strength) can be further used for the determination of the number of free carboxyl groups on the surface of PMAT-encapsulated NPs of different size and shape, at least within a certain size range, from a few nanometers to tens of nanometers. These findings become very important for studying the modification of PMAT-encapsulated NPs with different nonionic groups that are widely used for improving biocompatibility of NPs, such as polyethylene glycols, alkynes, etc. However, it should be pointed out that, in order to preserve complete dissociation of carboxyl groups for such study, one should use solutions with a pH value far above the isoelectric point of the covering polyelectrolytes (highly basic in the case of carboxyl groups).

Figure 5. Scheme for the possible arrangement of the PMAT molecules on {100} surface of zincblende ZnS. Blue spheres with black dots represent Zn atoms and hexanethiol bound to Zn, respectively (view from above). Colored wavy lines represent PMAT chains, and open circles are C12H25 alkyl branches.

Since the interaction of PMAT alkyl branches with hexanethiol bound to the surface Zn atoms is mainly governed by the van der Waals interaction, there should exist a certain distance between PMAT alkyl branches and hexanethiol molecules that provides efficient van der Waals (vdW) interaction. It is reasonable to assume that efficient vdW interaction requires a single MAT alkyl branch (open circle) positioned between two neighboring surface thiol molecules (black points), as depicted in Figure 5. One should also take into account the total length of a single MAT unit (∼5 Å, four C−C bonds taking into account the chain bending). After the comparison of this value with the lattice parameter of the ZB phase (a = 0.5401 nm), one may conclude that, for large-area NCs, there is no more than one alkyl branch from the same PMAT molecule per given single ZnS cell. However, another alkyl branch from the second PMAT molecule can fit into the same cell, thus giving, in total, one MAT unit per single Zn atom. Larger ratio of MAT units per single Zn atom for larger NPs with a surface area of >150 nm2 could be explained by the 1959

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type, pH, and ionic strength) is almost constant and independent of the size, shape, and chemical composition of cores. The average value of ζ = −35 mV at pH 9.5 and 5 mM ionic strength for PMAT-encapsulated NCs can be used as a reference point for the determination of the surface density of carboxyl groups upon PMAT modification with nonionic groups.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b04602. Protocol for the synthesis of 4-(fluorescein-6-carboxamido)-butylammonium chloride, TEM images and size histograms for all studied NCs, gel-electrophoresis results, protocol for the synthesis, and determination of the molar absorption coefficient of ZnSe QDs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support from Chemreagents Program. V.S. and M.K. acknowledge the financial support from the National Academy of Sciences of Belarus (3.1.03 “Convergence”).

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Figure 6. Zeta potential of different PMAT-encapsulated NPs as a function of (a) the surface area and (b) the number of carboxyl groups per single NP. Black dots represent CdSe/ZnS QDs#1−4 from Table 1, red dots represent CdS/ZnS QDs#5 and QDs#6, blue dot represents ZnSe/ZnS QDs#7, and green diamond represents CdS/ ZnS NRs#1. All zeta potentials were measured in a carbonate− bicarbonate buffer with pH 9.5 and 5 mM ionic strength. Solid curves are the linear fits of experimental data.

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CONCLUSION Labeling of amphiphilic polymer poly(maleic anhydride-alt-1tetradecene) (PMAT) with a controlled amount of dye molecules allows one to determine the polymer coverage of AIIBVI core−shell nanocrystals of various size, shape (dots, rods) and chemical composition (CdSe/ZnS, CdS/ZnS, ZnSe/ ZnS). The amount of polymer molecules attached to the surface of nanocrystals grows linearly to the surface area calculated from the average size of core−shell nanoparticle. However, the surface area per single MAT unit or equivalent average surface concentration of MAT units exhibits highly nonlinear character. For large nanocrystals, this behavior is likely due to the strong competition between PMAT molecules for surface binding sites represented by Zn-thiol complexes. On the other hand, in the case of small nanocrystals, incomplete surface coverage with PMAT is observed probably due to the faceted crystalline nature of ZnS surface with sulfur-terminated facets free from bound thiols. Such incomplete coverage may be the reason for the known limited chemical stability of AIIBVI luminescent QDs encapsulated with PMAT. For all types of nanocrystals (NCs) studied, the zeta potential (ζ) that is measured under fixed conditions (buffer 1960

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DOI: 10.1021/acs.langmuir.5b04602 Langmuir 2016, 32, 1955−1961