ZnS quantum dots with biocompatibility

Journal of Colloid and Interface Science 351 (2010) 1–9

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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis

In situ synthesis of highly luminescent glutathione-capped CdTe/ZnS quantum dots with biocompatibility Ying-Fan Liu a,b, Jun-Sheng Yu a,* a b

Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China Zhengzhou University of Light Industry, Zhengzhou 450002, PR China

a r t i c l e

i n f o

Article history: Received 29 January 2010 Accepted 17 July 2010 Available online 23 July 2010 Keywords: Semiconductor nanocrystals CdTe/ZnS Biocompatibility Photoluminescence Lifetime

a b s t r a c t This paper focuses on the in situ synthesis of novel CdTe/ZnS core–shell quantum dots (QDs) in aqueous solution. Glutathione (GSH) was used as both capping reagent and sulfur source for in situ growth of ZnS shell on the CdTe core QDs. The maximum emission wavelengths of the prepared CdTe/ZnS QDs can be simply tuned from 569 nm to 630 nm. The PL quantum yield of CdTe/ZnS QDs synthesized is up to 84%, larger than the original CdTe QDs by around 1.7 times. The PL lifetime results reveal a triexponential decay model of exciton and trap radiation behavior. The average exciton lifetime at room temperature is 17.1 ns for CdTe (2.8 nm) and 27.4 ns for CdTe/ZnS (3.7 nm), respectively. When the solution of QDs is dialyzed for 3 h, 1.17 ppm of Cd2+ is released from CdTe QDs and 0.35 ppm is released from CdTe/ ZnS. At the dose of 120 lg/ml QDs, 9.5% of hemolysis was induced by CdTe QDs and 3.9% was induced by CdTe/ZnS QDs. These results indicate that the synthesized glutathione-capped CdTe/ZnS QDs are of less toxicity and better biocompatibility, so that are attractive for use in biological detection and related fields. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Semiconductor quantum dots (QDs) have been widely studied as fluorescence probes in cell imaging and biological labeling [1–6] due to their optical and electric properties. Compared to organic fluorescence molecules, quantum dots present a series of excellent optical properties, including size-dependent tunable photoluminescence, broad excitation spectrum, narrow emission bandwidth and high photochemical stability. Besides, these common optical properties, quantum dots as biological markers are expected to possess not only high photoluminescence quantum yields (QYs), but also good biocompatibility. In the last two decades, great efforts have been made to synthesize highly fluorescent II–VI semiconductor QDs. For example, high fluorescence CdSe/CdS and CdSe/ZnS QDs have been synthesized by organometallic method and used as biological probes [7]. However, when these hydrophobic QDs are dispersed in aqueous environments for biological labeling, it is necessary to transfer these QDs from organic phase to aqueous solution through a post-treatment step, which will lead to remarkable decrease of photoluminescence quantum yields of QDs. In recent years, the direct preparation of hydrophilic QDs has been paid more attention because water-soluble QDs can be more convenient for their applications in biology labeling than hydrophobic QDs. The photoluminescence quantum yields of CdTe * Corresponding author. Fax: +86 25 3317761. E-mail address: [email protected] (J.-S. Yu). 0021-9797/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2010.07.047

QDs synthesized previously in the aqueous phase are usually only 3–10% [8,9]. The low quantum yields of QDs in the aqueous phase are attributed to defects and traps on the surface because the surface of a nanocrystal is made up of atoms that are not fully coordinated. To obtain highly fluorescent QDs, the selection of capped reagents is an important method. For example, thioglycolic acid (TGA) stabilized CdTe QDs present as high as 60% of the photoluminescence quantum yield in the aqueous phase [10,11]. Glutathione (GSH) has also been used as a capping agent for synthesis of CdTe QDs [12–14]. Because glutathione is an important water-phase antioxidant and essential cofactor for antioxidant enzymes, GSH-capped QDs were found to be more biocompatible than other water-soluble QDs. However, TGA- or GSH-stabilized CdTe QDs would be in part dissociated in biological environments due to the irradiation of excitation light, especially for application in the long-term imaging in living body. The dissociation of Cd2+ from CdTe QDs cannot only do harm to cells [15], but also weaken the luminescence intensity of the QDs due to their dissociation. Recently, several CdTe/CdS core–shell QDs with high fluorescence have been successfully synthesized by the epitaxial growth technique of inorganic shell [13–18]. These aqueous CdTe/CdS QDs with core–shell structure are commonly prepared by illuminating [16,17], microwave irradiation [18], ultrasonic [19], selective photochemical etching [20] and refluxing at low temperature [21] approaches. The growth of inorganic CdS shell on the surface of CdTe core QDs eliminates efficiently the surface dangling bonds and defects, resulting in forming core–shell structure of

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nanocrystals with high luminescence quantum yields (QYs usually up to 50–80%). Although these CdTe/CdS core–shell QDs will still maintain high fluorescence intensity even in biological environment and under continuous excitation, the core–shell QDs with a CdS shell may be also cytotoxic due to the toxicity of Cd2+ released from their surfaces of QDs. To obtain QDs with biocompatibility, the new core–shell CdTe/ZnS QDs [22] have been developed through organometallic synthetic approaches. In comparison with CdTe/CdS QDs, these CdTe/ZnS QDs evidently suppress the toxicity of the QDs itself in biological environments due to ZnS shell on the surface. Although the synthesis of QDs with a ZnS shell in the organic phase is an efficient route, rigorous experiment conditions such as high temperatures, a great amount of organic and toxic reagents are normally required. Furthermore, for bio-applications, these nonaqueous CdTe/ZnS QDs have to be transferred to aqueous solution by the exchange of ligands, which not only increases complexity in manipulation, but also decreases markedly PL quantum yields of QDs. The direct preparation of highly luminescent CdTe/ ZnS core–shell QDs has not previously been reported in aqueous solution. In this paper, we describe a novel strategy of in situ synthesis of core–shell CdTe/ZnS QDs in aqueous solution. Glutathione (GSH) is used as both capping reagent and sulfur source for the growth of ZnS shell on the CdTe core. The existence of a ZnS shell on the surface of CdTe core has been confirmed by XRD, XPS and EDX spectroscopy. The maximum photoluminescence quantum yield of CdTe/ZnS core–shell QDs synthesized is up to 84% refluxing for 10–20 min at 100 °C. The maximum emission wavelengths of asprepared CdTe/ZnS core–shell QDs can be simply tuned from 569 nm to 630 nm by varying pH values and reaction time at 100 °C. 0.35 ppm of cadmium ions from the dialysis solutions of CdTe/ZnS QDs are detected by atomic absorption spectroscopy (AAS), whereas 1.17 ppm of cadmium ions from TGA-CdTe QDs are found under the same dialysis condition. These results indicate that as-prepared CdTe/ZnS QDs can release less Cd2+ ions than CdTe QDs. To further explore GSH-CdTe/ZnS QDs biocompatibility, hemolysis assay has been carried out by the incubated mixture of QDs and erythrocyte from rabbit together for 3 h at 37 °C. As the final dose of QDs is 120 lg/ml, hemolysis induced by GSH-stabilized CdTe/ZnS QDs is only 3.9%, whereas TGA-stabilized CdTe QDs may induce higher hemolysis of 9.5%. Hemolysis data suggest that GSH-CdTe/ZnS QDs are more biocompatible than TGA-CdTe QDs. The new CdTe/ZnS core–shell QDs are expected to find wide applications in live cells, in vivo imaging and diagnostics. The synthetic route of glutathione-capped CdTe/ZnS QDs presented is simple, friendly-environmental and cost-effective compared with the conventional organometallic approaches. 2. Materials and methods 2.1. Materials Thioglycolic acid (TGA, 99%), NaBH4 (96%), tellurium powder (99.8%) and reduced glutathione (GSH, 99%) were obtained from Sigma, Inc. Na2HPO412H2O, KH2PO4, NaCl, and CdCl2 were obtained from Shanghai Chemical Reagents Company. Ultrapure water with 18.2 MX/cm was used in all syntheses. 2.2. Synthesis of TGA-stabilized CdTe QDs The synthesis of CdTe QDs was performed according to the references with some modification [23,24]. Typically, CdTe NCs were prepared by using the reaction between Cd2+ and NaHTe solution. Under vigorous stirring, the oxygen-free 0.1 mmol of NaHTe solution was prepared by the reaction of Te and NaBH4 in aqueous

solution. The concentration of Cd2+ and the molar ratio of Cd2+:HTe:TGA were set as 4 mM and 1:0.25:1.5, respectively. Under a robust flow of nitrogen, 91.3 mg (0.4 mmol) of CdCl22.5H2O was dissolved in 100 ml of Milli-Q water, and 44 ll (0.6 mmol) of pure TGA was added. Dropwise addition of 0.5 M NaOH was then used to adjust the pH to 11 under vigorous stirring. The resulting mixture solution between Cd2+ and NaHTe was heated to 100 °C under open-air conditions and refluxed 30 min for controlling the size of QDs. 2-Propanol was added to the as-prepared CdTe QDs colloid solution. CdTe QDs were precipitated from the solution and collected by centrifugation. Then, the precipitate was redissolved in proper Zn2+ and GSH solution, and the process of preparation was repeated to remove excess Cd2+ on surfaces of CdTe QDs. Finally, the obtained CdTe QDs were dried at room temperature under vacuum.

2.3. Synthesis of GSH-stabilized CdTe/ZnS QDs A typical synthesis of CdTe/ZnS core–shell nanoparticles is described hereafter. The 40 mg of as-prepared CdTe sample was added to the 50 ml solution (pH = 8) containing 1 mmol/l ZnCl2 and 4 mmol/l GSH. The solution was heated to 100 °C under open-air conditions and refluxed from 10 min to 2 h for controlling the sizes of core–shell QDs. Aliquots of the reaction solution were removed at regular intervals for UV absorption and PL experiments. This method is simple and the quantities can be easily scaled. Samples were precipitated by 2-propanol and dried in a vacuum oven for X-ray diffractometer (XRD) and X-ray photoelectron spectroscopy (XPS) characterization. Transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), and energy dispersive X-ray spectroscopy (EDX) samples were prepared by dropping the aqueous CdTe/ZnS solution onto carbon-coated copper grids with the excessive solvent evaporated.

2.4. Hemolysis assay This method was modified from a published procedure [25]. In the studies, the blood was drawn from rabbit into an evacuated siliconized glass tube and stirred to remove fibrinogen. The blood was diluted with an isotonic phosphate buffer (PBS) solution with pH 7.4 and centrifuged at 2500 rpm for 15 min and the supernatant was discarded. The PBS consisted of Na2HPO412H2O (7.95 g), KH2PO4 (0.76 g), NaCl (7.20 g), and distilled water (1000 ml). The erythrocytes were washed until supernatant was clear, and the packed cells were resuspended in PBS buffer solution (pH 7.4) to form 2% red blood cells. The stock dispersion was stored in a refrigerator for a maximum of 48 h and its stability was checked by photometric monitoring. Two kinds of powder QDs samples through the purified process were redissolved in PBS buffer solution for hemolytic assay. Two milliliters of the resultant erythrocyte suspension, TGA-capped CdTe or GSH-capped CdTe/ZnS QDs at different final doses (0, 40, 80, 120, 160 and 200 lg/ml) and appropriate amounts of phosphate buffer (the total volume 4 ml) were mixed and incubated together for 3 h at the 37 °C in an orbital shaker. The mixtures were centrifuged at 2500 r/min for 10 min and the supernatants were taken out. The degree of hemolysis was determined by measuring the absorbance of the supernatant at 540 nm and the absorbance of the QDs was subtracted out. The absorbance of the control group was used as the blank. Isotonic phosphate buffer (PBS) solution and deionized water were used as negative and positive controls, respectively. The absorbance value accounted for the amount of hemoglobin release induced by QDs. The hemolysis was calculated based on the average of three replicates.

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Hemolysis ¼

OD ðtestÞ  OD ðnegative controlÞ  100 OD ðpositive controlÞ  OD ðnegative controlÞ

2.5. Characterization UV–vis absorption and photoluminescence (PL) spectra were measured at room temperature with a Shimadzu 3600 UV–visnear-IR spectrophotometer and a NF920 fluorescence spectrometer, respectively. PL spectra were taken at the excitation wavelength kex = 475 nm for photoluminescence quantum yields (QY). Estimates of the QY were obtained by comparing the area of the fluorescence spectrum from Rhodamine 6G in ethanol (kex = 475 nm, QY: 0.95) [9]. Concentrations were adjusted such that both reference and QD samples gave optical densities of 0.02–0.05 at the excitation wavelength. Time-resolved luminescence measurements were carried out on a NF920 spectroflurometer with nanosecond flashlamp as light source. Slit widths of monochromators of both excitation and emission channels were set to 12 nm. Decay kinetics were recorded on m = 2049 channels. Typically, 3000 counts in a peak channel (CPC) of the decay curve were collected. Ludox was applied for PL lifetime measurement in order to eliminate the influence of light scattering (i.e., excitation and emission). Powder XRD measurement was taken on a Philips X’Pert PRO X-ray diffractometer. High-resolution transmission electron microscopy (HRTEM) was performed on a Philips FEI Tecnai G2 20 S-TWIN. X-ray photoelectron spectroscopy (XPS) was investigated by using a VG ESCALAB MK II spectrometer with a Mg Ka excitation. Binding energy calibration was based on C 1 s at 284.6 eV. An atomic absorption spectrometer (AAS) spectrum was measured using a Japan Hitachi-180-80 to analyze the elemental chemical composition of the QDs. All the samples were prepared by dissolving the QDs in 1% of HNO3 solutions. 3. Results and discussion 3.1. Synthesis of CdTe/ZnS QDs It has been reported that CdTe and CdSe QDs can induce cell death due to the release of Cd2+, whereas the CdSe/ZnS QDs present less cytotoxic due to the release of low Cd2+ concentration (<5 nM) which is confirmed by a Cd2+-specific cellular assay [26].Water-soluble CdTe/CdS QDs with high fluorescence synthesized recently are also cytotoxic due to the release of Cd2+ in biological environments. These QDs might not be suitable in long-term bio-application, especially in vivo. To overcome these shortcomings, we have developed a new route of CdTe/ZnS QDs synthesized in aqueous solution. In the route, the CdTe QDs were firstly synthesized with TGA as the stabilizer in aqueous solution, then GSH-stabilized CdTe QDs were prepared using TGA-capped CdTe QDs by the exchange of ligands GSH with TGA in solution. Finally, as-prepared GSHcapped CdTe QDs are used as core template, highly fluorescent CdTe/ZnS QDs are obtained through in situ growth of ZnS shell on the surface of CdTe core in alkaline solution containing Zn2+ and GSH at 100 °C water-bath (see Section 2.3). Fig. S1 shows the evolution of the luminescence images in situ growing process of ZnS shell on the CdTe core illuminated under room nature light. 3.2. Optical properties of CdTe/ZnS QDs Fig. 1 presents typical evolutions of both absorption and photoluminescence spectra of GSH-capped CdTe/ZnS QDs prepared in the aqueous phase. Although successfully producing CdTe dots with a wide range of sizes, we here focus on one particular sample of CdTe core with emission peaks at 540 nm and study the

3

evolution of the optical and structural properties as a function of ZnS coverage. The size and concentration of the core CdTe NCs are 2.8 nm and 2  106 mol l1, respectively, which is determined by using the empirical formula of the NCs [27]:

A ¼ ebc; e ¼ 10043 ðDÞ2:12 ; D ¼ ð9:8127  107 Þk3  ð1:7147  103 Þk2 þ ð1:0064Þk  ð194:84Þ: Here A is the absorbance at the peak position of the first exciton absorption peak for a given sample, c is the molar concentration (mol/l) of the NCs of the same sample, b is the path length (cm) of the radiation beam used for recording the absorption spectrum, D is the diameter of the NCs, and k (nm) is the wavelength of the first excitonic absorption peak of the corresponding sample. Heating of mixture solution of glutathione, Zn2+ and core CdTe NCs results in a gradually red shift of the maximum absorption and PL emission wavelength of the solution system (Fig. 1), which implies that a ZnS shell is slowly growing in situ on the CdTe core. With the reflux proceeding, the excitonic absorption peak of nanoparticles shifts to the longer wavelengths (lower energies) from 514 nm to 576 nm as the NCs grow to larger size. This result is agreed with the consequence of the quantum confinement effect. The corresponding PL emission wavelengths and QYs of the CdTe/ZnS NCs are 569 nm 84%, 578 nm 82%, 584 nm 75%, 596 nm 61%, 604 nm 40%, and 609 nm 29%, respectively. At first refluxing 10 min, the best photoluminescence QY (84%) of the CdTe/ZnS NCs obtained is larger than that of the original CdTe core NCs by around 1.7 times. At the same time, the size of the core–shell NCs increases to 3.7 nm (core 2.8 nm), which indicates that the thickness of ZnS shell is about 0.9 nm. After refluxing for 20 min, with photoluminescence wavelength increasing, the QY of CdTe/ ZnS decreases to 82%. Further heating to 30 min, the increasing rate of photoluminescence wavelength decreases and the brightness of the NCs with QY of 75% also declines. Finally, during refluxing 2 h, the emission wavelength of the QDs system is up to 609 nm, but the QY decreases to only 29%. These results indicate that under refluxing, the NCs are grown to their final size, and the fluorescence of the core–shell CdTe/ZnS NCs can be tuned in color with the refluxing time. As shown in the Fig. 1, the excitonic peak position shifts to longer wavelengths and the photoluminescence intensity first increases, and then declines, as the thickness of the ZnS shell increases. The increase is due to in situ the formation of ZnS shell resulting in the efficiently diminishing of the surface defects of core NCs and the lowering of confinement energy of exciton after capping core NCs with higher band gap shells [16–21] and the decline is believed to be a consequence of increased strain in the shell. Interfacial strain will play an important role which is from the large lattice mismatches between the CdTe core and ZnS shell (lattice parameters for CdTe c = 6.477 Å, and for ZnS c = 6.257 Å) [28]. On the other hand, it can be seen in Fig. 1 that the sizes of nanoparticles grow in aqueous solution accompanied by a clear the PL full width at half maximum (FWHM) narrowing. FWHM of the PL peak quickly changes from 40 nm to 36 nm during refluxing for 10–30 min. While continuing to prolong the reaction time, the FWHM restarts to broaden slowly from 36 nm to 40 nm, then to 46 nm during refluxing for 2 h. The narrow FWHM of the PL peak reflects the narrow particle size distribution, which is an efficient method to investigate the size focusing. Fig. 1 shows that with heating of mixture solution of glutathione, Zn2+ and core CdTe NCs, the PL efficiency of QDs system is greatly improved. The reason might be that the heating causes the slow decomposition of partial GSH with the release of sulfur to form in situ a ZnS shell around the CdTe core, resulting in the enhancement of the PL efficiency. The formation of a ZnS shell also greatly inhibits further oxidation of CdTe QDs. The more important is that when GSH is used as the S precursor, the release rate of

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A

B

0.5

g

b

80000

λ FWHM 540 nm 40 569 nm 36 578 nm 36 584 nm 36 596 nm 40 604 nm 42 609 nm 46

Intensity (a. u.)

Absorbance

0.4

0.3

b

g

0.2

60000

40000

a

QY 50% 84% 82% 75% 61% 40% 29%

20000 0.1

0.0 300

a 0 400

500

600

700

500

Wavelength (nm)

600

700

Wavelength (nm)

Fig. 1. Typical temporal evolution of the absorption (A) and corresponding emission (B) spectra of CdTe/ZnS NCs at the excitation wavelength (475 nm). a stands for spectra of CdTe core QDs. b, c, d, e, f and g stand for spectra of CdTe/ZnS QDs obtained for refluxing 10, 20, 30, 60, 90, and 120 min, respectively.

3.3. Effect of pH In situ growing process of core–shell CdTe/ZnS NCs, we found that the pH value of the synthetic solution is an important factor for preparation of highly luminescent NCs in aqueous solution. In the current aqueous CdTe synthesis, the pH value of the solution containing Cd precursors and TGA ligands was generally fixed at 11.2–11.8 [8]. At such high pH, relatively strong luminescence intensities (QY 50%) of CdTe NCs were obtained by adjusting the ratio of Cd2+:HTe:TGA (1:0.25:1.5). However, it is different for in situ synthesizing of the CdTe/ZnS NCs due to Zn precursor alternating Cd precursor. In order to study the influence of pH value on the brightness and stability of the NCs, we selected out three kinds of pH values (pH = 8.0, 9.5 and 9.9). The experimental results show that the QYs of the as-prepared CdTe/ZnS NCs increase with lowering of the pH of the solutions from the pH 9.9 to 8.0, further lowering of the pH leads to little precipitations of CdTe/ZnS NCs. As shown in Fig. 2, at pH = 9.9, the PL wavelength of CdTe/ZnS increases rapidly with refluxing time, up to 630 nm after 2 h, whereas the PL QY increases slowly and the largest PL QY is only 65%. While lowering of the pH to 9.5, the corresponding PL QY increases and the largest PL QY reaches 77%. When pH = 8.0, the best PL QY (84%) is obtained refluxing for 10 min, it is possibly owing to the formation of a better surface structure of CdTe/ZnS NCs under this synthetic conditions. The influence of pH perhaps is attributed to the formation of different Zn–GSH complex at certain pH values. Zn2+ can react with GSH to form Zn–GSH or Zn(GSH)2 complexes in various pH values, respectively. As a result of reaction balance, monothio Zn–SR complex existing in precursors directly led to increasing concentration of free Zn2+ ions, which would play an important role in the forming ZnS shell on the surface of a CdTe core, resulted in synthesizing of high luminescent CdTe/ZnS NCs. 3.4. Effect of ratio of Zn/GSH We note that the ratio of Zn/GSH is another important factor that strongly influences the PL QYs of GSH-capped CdTe/ZnS

100

CdTe/ZnS pH 9.9 9.5 8.0

C

80

CdTe

QY (%)

elemental S is slow enough to maintain the concentration below the critical level for nuclei formation [29]. In contrast, directly injecting S2 into the reaction system makes a rapid reaction take place between S2 and Zn2+ ions, only leading to the formation of an unhomogeneous shell on CdTe surface and the single ZnS clusters, which limits PL enhancement [30].

B

60

A

40

20

0 520

560

600

640

Wavelength (nm) Fig. 2. The influence of various pH values on PL QYs of the GSH-stabilized CdTe/ZnS NCs obtained after refluxing for 10, 20, 30, 60, 90 and 120 min (from left to right), respectively. pH = 9.9 (A), 9.5 (B) and 8.0 (C). Synthesizing conditions for all QDs samples: [CdTe] = 0.8 mg/ml (kmax = 540 nm), [Zn2+] = 1 mM, GSH = 4 mM and T = 100 °C.

core–shell NCs. In order to observe the effect of the ratio of Zn/ GSH, we fixed the concentration of CdTe core, GSH and other conditions such as pH value and reaction temperature, but only changed the concentration of Zn2+. The experimental data (Fig. 3) show that the influence of the various ratios of Zn/GSH on PL QYs of the GSH-stabilized CdTe/ZnS NCs. If the ratio of Zn/GSH is 1:8 (Fig. 3A), the PL emission wavelengths and QYs of the NCs obtained are 568 nm 70%, 582 nm 58%, 593 nm 51%, and 613 nm 22%, after refluxing for 10, 20, 30 and 60 min, respectively. While the ratio of Zn/GSH is 2:8 (Fig. 3B), the PL emission wavelengths and QYs of GSH-capped CdTe/ZnS NCs obtained are 569 nm 84%, 578 nm 82%, 584 nm 75%, and 596 nm 61%, respectively. With increasing the concentration of Zn2+, i.e., as the ratio of Zn/GSH is up to 3:8 (Fig. 3C), after adding Zn2+ to the solution of CdTe core QDs, the PL QY of CdTe core template is evidently decreased, but the PL QY of the QDs system increases significantly with the reflux proceeding. These results indicate that the ratio optimum of Zn/GSH is 2:8 for obtaining CdTe/ZnS NCs emitting with PL QY 61–84% at room temperature. At [ Zn2+]/[GSH] = 2:8, the fluorescent emission peak appears in the orange–red window, and the maximum PL QY increases up to 84% at room temperature without any postprepar-

Y.-F. Liu, J.-S. Yu / Journal of Colloid and Interface Science 351 (2010) 1–9

B Zn/GSH A 1:8 B 2:8 C 3:8

80

C

QY (%)

60

A

40

CdTe

CdTe/ZnS

20

540

570

600

630

Wavelength (nm) Fig. 3. The influence of the ratios of Zn/GSH on PL QYs of the GSH-stabilized CdTe/ZnS NCs obtained after refluxing for 0, 10, 20, 30 and 60 min (from left to right) respectively. The ratios of Zn/GSH are 1:8 (A), 2:8 (B) and 3:8 (C), respectively. Synthesizing conditions for all QDs samples: [CdTe] = 0.8 mg/ml, GSH = 4 mM, pH = 8.0 and T = 100 °C.

ative treatment. Because Zn2+ could provide a constant rate of transport to the particles, it could be impossible to form compact shell at lower Zn/GSH ratio (1:8, lower concentration of Zn2+). On the contrary at higher Zn/GSH ratio (3:8), although ZnS shell on the surface of CdTe core can eliminate the traps on the surface of the NCs, excess Zn2+ could produce new surface defects. Thus, the appropriate ratio of Zn/GSH cannot only provide compact ZnS shell, but also efficiently eliminate the traps and defects on the surface of the NCs. 3.5. TEM, XRD, XPS and EDX characterization In order to confirm the formation of ZnS shell on CdTe core in aqueous solution, X-ray photoelectron spectroscopy (XPS), energy dispersive X-ray spectroscopy (EDX), and the powder XRD measurements were made. At the same time, aqueous CdTe/ZnS NCs were characterized by transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM). A full survey scan and Cd, Te, S photoelectron spectra of the CdTe and CdTe/ZnS are displayed in Fig. 4. Besides the Cd3d core levels, the spectra are dominated by the C1s and O1s signals stemming from the capping agent. XPS spectra of CdTe QDs in Fig. 4 show that Cd3d, Te3d and S 2p are 404.89, 411.68; 571.77, 581.91 and 161.88 eV, respectively, while the binding energies of Cd3d, Zn2p, Te3d and S 2p for CdTe/ZnS QDs are 404.55, 411.39; 1020.84, 1044.08; 571.25, 571.76, 581.46, 582.24 and 161.48 eV, respectively. It is evident that the binding energies of Te in CdTe/ ZnS QDs occurred to split, which is different of that of Te in CdTe QDs. The reason may be that Te atoms binds not only to Cd atoms but also to Zn atoms in CdTe/ZnS QDs. Compared with CdTe core, there are higher content of sulfur, while lower content of tellurium on CdTe/ZnS surfaces. The ratios of (S + Te)/Cd are 0.97 for CdTe and 1.44 for CdTe/ZnS, respectively. The binding energy of S 2p shifts blue 0.5 eV from CdTe to CdTe/ZnS QDs. These data of Xray photoelectron spectroscopy (XPS) provide the direct evidence of the formation of CdTe/ZnS QDs. The EDX spectrum in Fig. S2 shows the existence of Te, Cd, Zn and S in CdTe/ZnS sample. The sulfur peak at 2.3 keV and the Zn peak at 8.9 keV in EDX spectrum indicate the existence of a ZnS shell layer on the CdTe core. These results of the EDX spectrum further confirm the formation of the core/shell QDs. The powder XRD pattern for the CdTe and CdTe/ZnS NCs are depicted in Fig. 5. The XRD patterns obtained from CdTe and CdTe/

5

ZnS powders, which were precipitated from aqueous solution with an excess of 2-propanol and the precipitate was isolated by centrifugation and dried at vacuum. The characteristic zinc blend planes of 111, 220, and 311 locating at 24.40°, 41.60°, and 47.90° for CdTe and at 24.94°, 41.72°, and 48.76° for CdTe/ZnS in the 2h range of 10–60° have been observed. The position of the XRD peaks of CdTe cores matched well with those of bulk CdTe cubic structure (JCPDS NO. 15-0770). After growth of ZnS shell on CdTe core, peak position shifted to higher angles towards the positions of bulk ZnS cubic structure peaks (JCPDS NO. 05-0566), which is proven the formation of CdTe/ZnS. The TEM and HRTEM images in Fig. 6 show that the CdTe/ZnS NCs possess a good dispersed crystalline structure, and have a diameter of about 3.7 nm, consistent with the results calculated from the absorption spectrum. The structural characterizations show a continuous growth of the crystallographic planes without a distinct boundary at the core–shell interface. Selected-area electron diffraction patterns also reveal that the lattice structure of the resulting nanoparticles is cubic (zinc blende). 3.6. Time-resolved luminescence decay measurements Luminescence decay kinetics provides additional important information on the recombination of photoinduced carriers in the NCs. The PL decay is monitored at the peak of the emission with the excitation wavelength at 400 nm. Fig. 7 shows the time-resolved luminescence decay curves of the CdTe and CdTe/ZnS NCs with sizes of 2.8 nm and 3.7 nm. The decay curves for the TGA-capped CdTe NCs and GSH-capped CdTe/ZnS show multiexponential recombination kinetics and the v2 values in the range of 1.00–1.30, which has been frequently observed for different kinds of II–VI NCs [24,31–35]. The multiexponential model is described by the following equation: Fit = A + B1 exp(t/s1) + B2 exp(t/s2) + B3 exp(t/s3). The decay lifetimes obtained from the experimental data in Fig. 7 are s1 = 2.5 ns (Rel: 0.5%) s2 = 14.1 ns (39.0%), s3 = 30.8 ns (60.5%) for CdTe, and s1 = 5.0 ns (0.1%), s2 = 24.0 ns (66.7%), s3 = 42.0 ns (33.2%) for CdTe/ZnS, respectively. The shorter-lifetimes (2–6 ns) in the decay curves could be attributed to the popular intrinsic recombination of QDs [31]. The shorter luminescence lifetimes of CdTe and CdTe/ZnS QDs are 2.5 ns and 5.0 ns, respectively. The result implies that the popular intrinsic recombination of QDs changes greatly because of the formation of new ZnS shell. The longer lifetimes (10 ns or more) can be attributed to thermal detrapping of the electrons from the surface states to the conduction band since such thermal activation could enhance the lifetime at the band-edge emission. The formation of CdTe/ZnS QDs can result in high luminescence quantum yields possibly due to removal of surface defects or by quenching non-radiative exciton recombination routes, which is advantage of thermal activation of the electrons and enhance the lifetime. This was witnessed by the data of the longer lifetime for CdTe/ZnS QDs. An alternative approach to analyze the fluorescent lifetimes is to use a model where the time at which the emission intensity had decreased to 1/e of its initial value was used. This allowed us to obtain a singular decay parameter, which can be considered as an effective average lifetime (s1/e) [32,33], deducing it from the PL decay curves presented in Fig. 7. The corresponding average lifetimes (s1/e) of the CdTe with 2.8 nm and CdTe/ZnS with 3.7 nm are 17.1 ns and 27.4 ns, respectively. It is evident that the average PL decay times increase steadily with increasing size of the NCs, a tendency that is generally observed for II–VI NCs [32,36]. 3.7. The possible growth mechanism of CdTe/ZnS QDs Because of their small size, QDs still have many inhomogeneous defects existing on the surface, which can cause a non-radiative relaxation resulting in a low photoluminescence (PL) efficiency.

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Y.-F. Liu, J.-S. Yu / Journal of Colloid and Interface Science 351 (2010) 1–9

A

B

6000 Cd3d5/2

CdTe/ZnS

CdTe/ZnS

Cd3d3/2

5000

1000

4000

800

CPS

CPS

1200

CdTe

3000

S2p

600 CdTe

2000

400 1000 395

400

405

410

415

420

154

156

Binding Energy (eV)

158

160

162

164

166

168

170

Binding Energy (eV)

C

D 3300

Te3d5/2

Te3d3/2

CdTe/ZnS

Zn2p3/2

8200

Zn2p1/2

CPS

CPS

3200

3100

CdTe

8000

7800

3000

CdTe/ZnS 7600

2900

565

570

575

580

585

590

Binding Energy (eV)

1010

1020

1030

1040

1050

1060

Binding Energy (eV)

Fig. 4. XPS spectra of CdTe and CdTe/ZnS: (A) cadmium 3d, (B) sulfur 2p, (C) tellurium 3d levels of CdTe and CdTe/ZnS, and (D) zinc 2p levels of CdTe/ZnS. The ratios of (S + Te)/Cd of CdTe and CdTe/ZnS are from 0.97 to 1.44.

ZnS

(111)

CdTe-ZnS

(220)

Counts (a.u.)

(311)

CdTe

CdTe 10

20

30

40

50

60

2θ (degree) Fig. 5. XRD patterns of TGA-capped CdTe (bottom) and GSH-capped CdTe/ZnS (top) NCs. The standard diffraction lines of cubic CdTe and cubic ZnS are also shown for comparison.

Generally, the QDs with core–shell structure are formed through inorganic epitaxial growth, so that the inorganic shell could efficiently eliminate both the anionic and cationic surface dangling bonds. In the growth process of inorganic shell, molecular precursors of the shell play an important role in controlling the rates of nucleation and growth of nanocrystals in solution system. In this work, GSH-stabilized CdTe QDs whose surfaces absorb excess Zn2+ are prepared through the process of twice precipitation and

Fig. 6. TEM image of CdTe/ZnS samples taken from reaction solution after refluxing 10 min. Inset: HRTEM image and SAED pattern of the CdTe/ZnS NCs.

re-dissolution from TGA-capped CdTe QDs. Fig. 8A depicts a schematic illustration of the growth process of ZnS shell on the surface structure of CdTe QDs, and the possible growth mechanism is as follows: (1) the as-prepared TGA-capped CdTe QDs are precipitated

Y.-F. Liu, J.-S. Yu / Journal of Colloid and Interface Science 351 (2010) 1–9

Normalized Intensity

3000

a CdTe b CdTe/ZnS

2400

1800

1200

a

b

600

7

result, Zn2+ ions gradually occupies with cadmium vacancies on the surface of the core QDs. The CdTe QDs fully capped with Zn2+ and GSH are obtained as the molecular precursors for the growing core–shell structure QDs; and (5) the aqueous CdTe/ZnS QDs are obtained via in situ the growth of a ZnS shell on the surface of CdTe core with the decomposition of glutathione refluxing at 100 °C (Fig. 8B). The formation process of the core–shell CdTe/ZnS QDs is that Zn2+ ions gradually occupies with cadmium vacancies, and then ZnS shell grows on the surface of the core QDs, which is described as the mechanism of ‘occupying vacancy’ and in situ growing.

3.8. Hemolysis assay of CdTe/ZnS QDs

0 80

120

160

200

240

Time (ns) Fig. 7. Time-resolved luminescence of the CdTe (a) with 2.8 nm and CdTe/ZnS (b) QDs with 3.7 nm (kex = 400 nm). Decay parameters are s1 = 2.5 ns (Rel: 0.5%), s2 = 14.1 ns (39.0%), s3 = 30.8 ns (60.5%), v2 = 1.037 for CdTe, and s1 = 5.0 ns (0.1%), s2 = 24.0 ns (66.7%), s3 = 42.0 ns (33.2%), v2 = 1.060 for CdTe/ZnS NCs, respectively. The equation used to simulate luminescence decay curves: Fit = A + B1 exp(t/s1) + B2 exp(t/s2) + B3 exp(t/s3).

with 2-propanol, and the supernatant is decanted to remove the excess Cd2+ and TGA; (2) the CdTe QDs obtained by first precipitation are dissolved in alkaline solution containing Zn2+ and GSH, thus excess Zn2+ ions and GSH in solution would occupy vacancies on the surface of the CdTe Core; (3) the second precipitation is carried out with the same procedure to remove some remaining Cd2+ and TGA; (4) the precipitates of CdTe QDs obtained in twice are redissolved in alkaline solution containing Zn2+ and GSH, as a

Determination of hemolytic properties is one of the most common tests in studies of nanoparticle interaction with blood components [37–39]. Erythrocyte interaction with QDs is particularly important in the use of QDs for in vivo applications. Hemolysis is one of indicators of interaction and incompatibility of QDs with red blood cells [40]. Fig. 9 shows the results of our assay for determination of the hemolytic properties of TGA-CdTe and GSH-CdTe/ZnS QDs. In this commonly used protocol, the QDs were incubated in purified erythrocytes rather than whole blood, and inclusion of various anticoagulants, the blood was centrifuged to remove undamaged erythrocytes, and the percent hemolysis was determined by colorimetric detection of hemoglobin in the supernatant. As shown in Fig. 9A, with prolonged incubation duration of mixtures of QDs and erythrocyte from 1 h to 4 h at 37 °C, the hemolysis rates are increased from 4.6% to 19.4% for TGA-CdTe and from 0.2% to 10.7% for GSH-CdTe/ZnS QDs. The effect of doses of the QDs on hemolysis was also measured (Fig. 9B). These results show that higher hemolysis

Fig. 8. (A) The schematic illustration of the transformation of the surface structure of CdTe core QDs via twice precipitation [(1) and (3)] and twice dissolution [(2) and (4)]. I, CdTe QDs solution containing excess Cd2+ and TGA; II, TGA-stabilized CdTe QDs precipitates; III, CdTe QDs solution containing a few remaining Cd2+, excess Zn2+ and GSH; IV, GSH-stabilized CdTe QDs precipitates. V, CdTe QDs solution containing excess Zn2+ and GSH. (B) The scheme of the formation of CdTe/ZnS core–shell QDs.

Y.-F. Liu, J.-S. Yu / Journal of Colloid and Interface Science 351 (2010) 1–9

Percentage Hemolysis

A

20

B

CdTe-TGA

16

12

8

4

CdTe/ZnS-GSH

Percentage Hemolysis

8

20 CdTe-TGA

15

10

5 CdTe/ZnS-GSH

0

0 1.0

1.5

2.0

2.5

3.0

3.5

4.0

40

80

120

160

200

Concentration ( g/ml)

Time (hour)

Fig. 9. Time (A) and concentration (B) dependences of hemolysis induced by CdTe-TGA (up) and CdTe/ZnS-GSH QDs (down). (A) The concentration of QDs is 120 lg/ml and the incubated durations of QDs and erythrocyte are 1, 2, 2.5, 3, 3.5 and 4 h at the 37 °C, respectively. (B) The incubated time of QDs and erythrocyte together is 3 h at the 37 °C and the final concentrations of QDs are 40, 80, 120, 160 and 200 lg/ml, respectively (from left to right).

Table 1 The contents of cadmium and zincum ions from the dialysis solutions of the CdTe and CdTe/ZnS QDs for 3 h and 5 h. QDs

CdTe (3 h) CdTe (5 h) CdTe/ZnS (3 h) CdTe/ZnS (5 h)

Metal ions

50 mM PBS solution. These results suggest that as-prepared GSHCdTe/ZnS QDs have less toxicity and better biocompatibility. 4. Conclusion

Zn2+ (ppm)

Cd2+ (ppm)

0.69 2.50

1.17 2.70 0.35 1.31

occurred for the QDs with higher doses. The dose dependent hemolysis is better fitted by liner curve function y = 0.0936x  1.53 (R = 0.991) for CdTe QDs and y = 0.04475x  1.27 (R = 0.996) for CdTe/ZnS QDs, and the liner slope of CdTe/ZnS QDs is less than that of CdTe QDs. For the same conditions such as incubation time, incubation temperature (37 °C) and the dose of the QDs, compared with hemolysis of TGA-CdTe QDs, the percent hemolysis of the GSHCdTe/ZnS QDs is evidently decreased. For example, as the final concentration of QDs is 120 lg/ml, CdTe QDs induce over 9% of hemolysis when contact with erythrocyte for 3 h, whereas the hemolysis of CdTe/ZnS QDs is reduced and lower than 5%. These studies show that GSH-CdTe/ZnS QDs are of better biocompatibility. Therefore, hemolytic activity is highly dependent upon nanostructured material composition as well as its surface environments (e.g. charge, ligand).

In summary, a simple and friendly-environmental methodology is presented for in situ preparing highly fluorescent CdTe/ZnS NCs in the aqueous phase. GSH existed widely in the cell cytosol is an excellent stabilizer for the preparation of aqueous CdTe/ZnS owing to its better biocompatibility and easier thermal decomposition. The maximum emission wavelengths of the prepared CdTe/ZnS QDs can be simply tuned from 569 nm to 630 nm and the optimum PL QY is up to 84%. Fluorescence lifetime measurements reveal a triexponential decay model of exciton and trap radiation behavior. In comparison with CdTe QDs, the as-prepared CdTe/ZnS QDs are of lower cellular toxicity and better biocompatibility and photostability. These core–shell QDs are expected to have wide applications in vivo molecular imaging, bio-labeling and other related fields. The synthetic route presented can be easily extended to the large-scale, aqueous-phase production of core–shell NCs. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 20875045), the National Science Fund for Creative Research Groups (No. 20821063), and the National Basic Research Program of China (No. 2010CB732401).

3.9. Assay of Cd2+ released from CdTe and CdTe/ZnS QDs CdTe QDs is cytotoxic due to toxic Cd2+ released from their cores, whereas the CdSe/ZnS QDs present less cytotoxic due to the release of low Cd2+ concentration. In this work, to assess toxicity of as-prepared GSH-CdTe/ZnS QDs, we detected the Cd2+ and Zn2+ concentration of the dialysis solutions from CdTe and CdTe/ZnS QDs in PBS media by atomic absorption spectroscopic analysis (AAS), respectively. Firstly, the powder GSH-CdTe/ZnS and TGA-CdTe QDs samples of 22 mg were taken and dissolved in 50 ml of 50 mM PBS solution, respectively. Then, the each solution was dialyzed for 3 and 5 h by dialysis membrane (MW 3000). Finally, Cadmium and zincum ions from the dialysis solutions were determined by AAS. As shown in Table 1, Cd2+ ions found in the CdTe dialysis solution are 1.17 and 2.70 ppm for the dialysis of 3 and 5 h, respectively. Cd2+ ions are only 0.35 and 1.31 ppm, and Zn2+ ions are 0.69 and 2.50 ppm in CdTe/ZnS dialysis solution for the same time of dialysis. The experimental data indicate that the content of free Cd2+ ions released from the CdTe/ZnS QDs are lower than that of CdTe QDs in

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