Hydrothermal synthesis of GSH–TGA co-capped CdTe quantum dots and their application in labeling colorectal cancer cells

Colloids and Surfaces B: Biointerfaces 95 (2012) 247–253

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Hydrothermal synthesis of GSH–TGA co-capped CdTe quantum dots and their application in labeling colorectal cancer cells Yongli Yu a , Linru Xu a,1 , Jing Chen a , Huanyu Gao a , Shuo Wang b , Jin Fang b , Shukun Xu a,∗ a b

Department of Chemistry, Northeastern University, Shenyang 110819, PR China Department of Cell Biology, Key-lab of Cell Biology of Ministry of Public Health, China Medical University, Shenyang 110001, PR China

a r t i c l e

i n f o

Article history: Received 21 February 2012 Received in revised form 5 March 2012 Accepted 12 March 2012 Available online 28 March 2012 Keywords: CdTe QDs GSH TGA Immunolabeling Colorectal cancer cell imaging

a b s t r a c t We have successfully synthesized GSH and TGA co-capped CdTe quantum dots (QDs) with good biological compatibility and high fluorescence intensity. The effects of different reaction time, temperature, pH value, ligand concentration and the molar ratio of GSH/TGA were carefully investigated to optimize the synthesis condition. The optical properties of as-prepared CdTe QDs were studied by UV–visible absorption spectrum and fluorescence spectrum, meanwhile their structure and morphology were characterized using transmission electron microscope (TEM), Fourier transform infrared spectra (FT-IR) and X-ray powder diffraction (XRD). Compared with the CdTe QDs that are single-capped with either GSH or TGA, the GSH–TGA co-capped CdTe QDs demonstrated significantly improved fluorescence intensity and optical stability. In addition, GSH–TGA co-capped CdTe QDs were conjugated to amonoclonal antibody ND-1. The GSH–TGA co-capped CdTe QDs-antibody probe was successfully used to label colorectal cancer cells, CCL187, in vitro. © 2012 Elsevier B.V. All rights reserved.

1. Introduction In the past two decades, quantum dots (QDs), as a type of novel biochemical probe and sensor, have attracted widespread attention due to their ideal optical and chemical properties, such as large Stokes shifts, high brightness and photochemical stability [1–7]. Various synthesis strategies of QDs aiming to obtain nanoparticles within a relatively narrow size range have been described, including control of size and stability [8–10], size-selective precipitation [11,12], cages of molecular sieves [13], and varying starting pH [14] etc. Rapid synthesis of high quantum yield CdTe nanocrystals in the aqueous phase by microwave irradiation with controllable temperature [15] was also reported. A very new synthesis method for QDs was reported by Yang’s group [16], in which highly luminescent QDs were synthesized in a “conical flask”. Through a room-temperature N2 H4 -promoted strategy, water-soluble CdTe QDs were easily synthesized by a stepwise addition of raw materials in one pot. No preparation of precursors, pH adjustment, heating, and even N2 protection were required. Just by adjusting the feed ratio of the reagents, QDs of different sizes were readily obtained

∗ Corresponding author. Tel.: +86 2483681343; fax: +86 2483687673. E-mail addresses: [email protected] (L. Xu), [email protected] (S. Xu). 1 Present address: Institute of Microanalytical Systems, Chemical Laboratory Building, Room 101, Zhejiang University (Zijingang Campus), Hangzhou 310058, PR China. 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2012.03.011

within much shorter time than other conventional methods. More importantly, besides conventional thiol-ligands such as thioglycolic acid (TGA), 3-mercaptopropionic acid (MPA), 1-thioglycerol (TG), 2-mercaptoethylamine (MA), glutathione (GSH), and l-cysteine (LCS), this method also allows for the use of some reagents which could not be used in the conventional reflux methods because of their poor water-solubility, including 4-mercaptobenzoic acid (MBA), per-6-thio-␣-cyclodextrin (␣-CD-SH), and per-7-thio-␤cyclodextrin (␤-CD-SH). There has been a rapid growth in the development of QDs capped with thiol-ligands such as TGA, MPA, mainly due to the bright and photostable fluorescence of these QDs. Meanwhile, much attention has been focused on the applications of QDs in the fields of bio-analysis, environmental analysis and clinical or medical detection. However, the question on the potential cytotoxicity due to the inherent toxic chemical composition of these QDs, e.g. heavy metal, remains unanswered. Recently, it has been reported that this cytotoxicity problem could potentially be solved by using a proper stabilizer to modify the surface of QDs [17]. l-Cysteine, an aminophenol naturally present in human body without any cytotoxicity, is considered to be a suitable stabilizer. Fischer et al. [18] explored the possibility of using relatively inexpensive cysteine for the synthesis of QDs. It had been shown that cysteine could play a similar role as phytochelatins [19] in controlling the size of QDs in the synthesis process. The issue with the phytochelatins system is that isolation of peptides is often associated with high cost. Su et al. demonstrated the feasibility of utilizing

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cysteine-stabilized CdTe QDs for in vitro and in vivo fluorescent imaging [20]. Yan and co-workers [21] reported a simple method to prepare CdTe/Cd(OH)2 core/shell nanoparticles using l-cysteine as stabilizer. Another method developed for the preparation and purification of l-cysteine capped CdTe QDs [22] highlighted many advantages such as greatly shortened synthesis time, wide range of starting pH value, wide range of fluorescence emission wavelength. In addition, a special purification process was developed to remove excess Cd2+ in the QDs solution to reduce the cytotoxicity. A simple, green room temperature synthesis method was also developed for ZnSe QDs by using ascorbic acid as stabilizer [23], in which a facile “green”, one-pot synthesis of starch-capped CdSe QDs with an obvious quantum confinement effect was achieved via a novel non-organometallic mechanism [24]. Most recently, direct bioconjugation of CdS quantum dots with bovine serum albumin (BSA) was obtained using a one-step colloid chemistry method in aqueous condition at room temperature [25]. Essentially, the bioconjugates were generated with BSA as capping ligand for the nucleation as well as the stabilizer for CdS QDs which was formed from cadmium perchlorate and thioacetamide as precursors. The results clearly indicated that BSA was an effective nucleating and stabilizing agent for the colloidal CdS quantum dots. l-Glutathione, a tripeptide of human body without any cytotoxicity, was also used as the stabilizer for the preparation of QDS [26]. GSH stabilized CdTe QDs were directly prepared in aqueous solution and used both for the indirect immuno-labeling of fixed prostate cancer cells [27] and direct labeling of mouse T lymphocyte and spleen tissues [28]. Recently, the effects of QDs capped with different chiral forms of GSH on its cytotoxicity and induction of autophagy were examined [29]. Two different sizes of CdTe QDs coated with either l-GSH (l-GSH-QDs) or d-GSH (d-GSH-QDs) were shown to have both dose-dependent cytotoxicity and significantly increased levels of autophagic vacuoles. Interestingly, the activation of autophagy was chirality-dependent, with l-GSH-QDs being more effective than d-GSH-QDs. The optical performances of reductive GSH-stabilized CdSe QDs in water were studied with varying luminescence condition and sample ambience [30], and a model was built to fit the evolution curves to deduce the dynamics of the system. Different sizes of GSH-capped CdTe (GSH/CdTe) QDs have been prepared directly in aqueous solution and covalently conjugated with folic acid to be used for cancer cell imaging, as reported by Tang’s group [31]. This demonstrated the potential of CdTe QDs in the broad application as bio-labels. Hou’s group [32] developed a novel determination method for the potential visual fluorescence probe of As (III), based on the fact that emission of GSH/CdTe QDs is quenched effectively in the presence of As (III) because As (III) has a high affinity toward reduced-GSH to form As(SG)3. To the best of our knowledge, there has been no report on the synthesis of CdTe QDs using both TGA and GSH as stabilizers. In this work, GSH and TGA co-capped CdTe QDs with good biological compatibility and high fluorescence intensity were successfully synthesized. The effects of reaction time, temperature, pH value, ligand concentration and the molar ratio of GSH:TGA were carefully investigated. Compared to CdTe QDs single-capped with either GSH or TGA, the GSH–TGA co-capped CdTe QDs demonstrated significantly improved fluorescence intensity and optical stability. The as-prepared QDs were conjugated with monoclonal antibody ND-1 to form immuno probe. The QDs-antibody probe was successfully used to label colorectal cancer cells CCL187 through the specific reaction between ND-1 antibody and LEA antigen. Control experimental results indicated that the labeling of colorectal cancer cells by QDs-ND-1 was specific. Compared with other methods, the direct labeling procedure was much simpler and more selective. Our study provided a novel agent which potentially could be used to detect colorectal cancer cells in vivo with high sensitivity and

contribute to the development of a promising method for imaging, early diagnosis and even treatment for cancer. 2. Experimental 2.1. Materials All chemicals used in the experiments were commercially available without further purification. CdCl2 ·2.5H2 O, NaBH4 (96%), tellurium powder (99.999), CuCl2 ·2H2 O, and thioglycolic acid were purchased from National Medicines Corporation Ltd. of China, Shenyang. All the chemicals above were of analytical grade. Nethyl-N -[3-(dimethylamino) propyl]carbodiimide hydrochloride (EDC, 98%), N-hydroxysuccinimide (NHS, 98%) and glutathione were purchased from Acros Organics (USA). l-Glycine was purchased from Peking Dingguo Biology Techniques Company (China). Colorectal cancer cells CCL187 and monoclonal antibody ND-1 was supplied by China Medical University. Triple-distilled water was used throughout the experiments. 2.2. Preparation of GSH–TGA Co-capped CdTe QDs CdTe QDs stabilized by GSH and TGA were synthesized in aqueous solution based on a previous publication [7] with some modifications. In a typical synthesis, NaHTe solution was produced by the reaction of sodium borohydride with tellurium powder in an ice bath for about 3 h. 0.48 mmol GSH and 0.48 mmol TGA were added into nitrogen-saturated 0.02 mol L−1 CdCl2 aqueous solution (40 mL) at pH 9.0–10.0. The mixture was stirred under N2 for 20 min. Then freshly made NaHTe solution (0.25 mol L−1 , 0.8 mL) was quickly injected into the mixture. After the reaction lasted for another 20 min, CdTe precursors were obtained. Finally, the precursors were transferred to an autoclave, sealed, and hydrothermally treated at 140 ◦ C for different times to prepare different sizes of GSH–TGA–CdTe QDs. The final molar ratio of Cd2+ /Te2− /GSH–TGA was set to 1/0.25/1.2, giving a final concentration of CdTe QDs about 5.0 × 10−3 mol L−1 (as Te). 2.3. Characterization The absorption spectroscopy of the prepared CdTe DQs was measured with a 2100 spectrophotometer (Ruili, Peking, China) using a 1 cm cuvette. Fluorescence measurements were performed on a LS-55 luminescence spectrometer (PerkinElmer, USA). Fourier transform infrared spectra were collected with a Bruker Tensor 27 FT-IR spectrometer (Bruker, Germany) in KBr media. Powder Xray diffraction patterns of QDs were carried out using an X Pert Pro MPD X-ray powder diffractometer (PW3040/60, P Analytical B.V., Holand) with Cu K␣ radiation (40 kV,  = 0.15406 nm). The size and morphology of as-prepared QDs were characterized by a JEM2000EX transmission electron microscope (TEM, Jeol Ltd., Japan), using an accelerating voltage of 120 kV. An Olympus IX51 fluorescence microscope equipped with blue excitation was used to image the colorectal cancer cells. 2.4. Immuno-labeling of colorectal cancer cells The as-prepared CdTe QDs were first conjugated with monoclonal antibody ND-1, an antibody recognizing the large external antigen (LEA) receptor on the CCL187 cell membrane, using NHS and EDC as cross-linkers. Typically, 50 ␮L of 0.1 mg mL−1 antibody solution in 10 mM PBS of pH 7.4, 50 ␮L of 0.1 mg mL−1 NHS and 50 ␮L of 0.1 mg mL−1 EDC were mixed with 80 ␮L of as-prepared CdTe in PBS buffer (pH 7.4), followed by incubation for 1.5 h at 37 ◦ C in a reciprocating oscillator. The colorectal cancer cells were cultured (37 ◦ C, 5% CO2 ) on glass chamber slides in RPMI 1640 media

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Fig. 1. TEM image (left) and XRD pattern (right) of as prepared CdTe QDs.

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found on the surface of the CdTe quantum dots, which made it feasible for antibody conjugation and subsequent cell labeling.

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The morphology and size of the GSH–TGA co-capped CdTe QD synthesized with pH 9 at 140 ◦ C for 1.5 h were characterized by TEM as shown in Fig. 1(left). The as-prepared QDs were spherical shaped with an almost uniform diameter of about 3 nm, which is supported by the result of 3.26 nm calculated by a reported equation [33]: D = 9.8127 × 10−7 3 − 1.7147 × 10−3 2 + 1.0064 − 194.84, where D (in nm) is the size of the QDs,  is the absorption peak position (in nm). X-ray diffraction (XRD) was used to characterize the crystal structure of the as-prepared CdTe QDs, and the XRD patterns are illustrated in Fig. 1(right). The three diffraction peaks of CdTe QDs at 24.59◦ , 40.86◦ , and 47.39◦ can be indexed to the (1 1 1), (2 2 0), (3 1 1) planes of cubic CdTe lattice (JCPDS card: 01-075-2086). The infrared spectra (IR) of TGA, GSH and the TGA–GSH costabilized CdTe QDs are shown in Fig. 2(a)–(c), respectively. The most pronounced IR absorption bands of the prepared CdTe QDs occur at 3410 (NH ), 2930 cm−1 (CH2 ), 1590 cm−1 (as − ),

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here were of a diameter of about 3.3 nm, prepared with pH 9 at 140 ◦ C for 1.5 h, and the fluorescence spectrum was measured with 350 nm excitation, whereas the excitation spectrum was monitored by 530 nm emission. These characteristics suggested that the products could be applied in bio-analysis and biological labeling indirectly. Meanwhile, the wavelength of the fluorescent emission peak ranged from 510 nm to 640 nm as shown in Fig. 3(right). Accompanied with the increase of emission wavelength, the emission colors varied from blue to red. Twelve QDs solutions with different emission wavelengths were prepared and characterized by the fluorescence spectrometer. The emission peaks of the five typical prepared CdTe QDs were normalized and shown in Fig. 3(right), with corresponding photographs taken under UV light (365 nm), which were prepared with pH 9 at 140 ◦ C for (from left to right) 1.0, 1.5, 2.0, 2.5 and 3.0 h, respectively.

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containing 10% fetal bovine serum and 1% penicillin/streptomycin overnight in a culture box. Then the colorectal cancer cells were incubated with antibody-functionalized CdTe QDs at 37 ◦ C for 1.0 h. After being washed with PBS for four times, the cells were imaged using an inverted fluorescent microscope (Olympus IX51, blue excitation). In a control experiment, CdTe without antibody conjugation was allowed to interact with colorectal cancer cells directly using above method.

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3.2. Optical properties of the as-prepared CdTe QDs The prepared GSH–TGA co-capped CdTe QDs have excellent optical properties such as broad UV absorption and excitation spectra, strong fluorescence intensity, narrow emission peak with the FWHM of 40 nm, as shown in Fig. 3(left). The presented QDs

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Fig. 3. Fluorescence excitation (a), emission (b) and absorption (c) spectra as well as the normalized emission peaks and corresponding photographs taken under UV light (365 nm) of as prepared CdTe QDs.

3.3. Optimization of synthesis conditions The pH of the reaction solution has a great influence on the surface nature and optical property of GSH–TGA co-capped CdTe QDs. With a molar ratio of ligands/Cd as 1.2:1, reaction temperature at 140 ◦ C and reaction time of 1.5 h, the effect of pH values of 8.0–11.0 on the QDs’ fluorescence spectra were studied. As shown in Fig. 4(left), with the increase of pH value, the spectrum of the QDs showed marked red shift of 24 nm, meanwhile the intensity increased first and decreased later, with the maximum intensity at pH 9.0. We proposed that this phenomenon could be attributed to the effect of pH values on the S H bond strength on the QDs’ surface. With the increase of pH value, the binding force increased and the capped Cd2+ increased, resulting the formation of larger QDs. Whereas when the pH value is too high, the formation of Cd(OH)2 could interfere with QDs’surface nature, leading to the decline of fluorescence intensity. So, pH 9 was chosen in the following experiments. Reaction temperature, which controls the nucleation rate, is also an important factor for QDs quality. Compared with the bathing method, the hydrothermal synthesis carried out in agitated reactor with higher temperature which offers several clear advantages, such as short reaction period, good uniformity and strong fluorescence of the products. The fluorescence spectra (em = 514 nm, 518 nm, 538 nm, 588 nm, 609 nm) of the QDs prepared at different hydrothermal temperature (100 ◦ C, 120 ◦ C, 140 ◦ C, 160 ◦ C, 180 ◦ C)in 1.5 h are shown in Fig. 4(right). At relatively low temperature between 100 and 120 ◦ C, the QDs grew very slowly and the maximum emission wavelength only showed a 4-nm red shift. With moderate increase in temperature, the QDs started to grow quickly, indicating the crystals’ growth requires a relatively higher temperature. However, when the temperature was too high, the crystals

grow too fast to allow sufficient reaction with stabilizers. In addition, formation of Cd(OH)2 precipitation formed led to the decline of fluorescence intensity. Based on the results obtained, we chose 140 ◦ C as the optimum temperature. Reaction time is another important factor to consider for the optimal synthesis of high quality QDs. Fig. 4(middle) shows the fluorescence spactra (em = 511, 538, 560, 589, 615, 632, 635, 641 nm respectively) of the QDs prepared at 140 ◦ C with different hydrothermal reaction time. When the reaction was carried out for only 0.5 h, there was not any fluorescence because the hydrothermal time was too short to form nano-crystals. Whereas the fluorescence intensity increased with the prolonging of the reaction time, accompanied with obvious red shift of the maximum emission wavelength, reaching the maximum intensity with a reaction time of 1.5 h. Then the fluorescence intensity decreased gradually with further prolonged reaction time, most likely due to the oxidation of the stabilizer molecules which resulted in reduced stabilization effect of the ligands and increased surface defects of QDs. In addition, it was clear that there are the red shift of emission maximum position and spectral broadening with increasing pH, reaction time or temperature, probably due to the increase in size and/or aggregation of the QDs [34]. The amount of stabilizer also influences QDs’s fluorescence property. As shown in Fig. 5(left), with the increase of the amount of stabilizers used in the reaction, the fluorescence spectrum showed red shift of emission wavelength due to increase of size of the particles. While the intensity was trending up initially with more stabilizers, it trended downward later on the maximum intensity obtained with a molar ratio of ligand/Cd at 1.2:1. One plausible explanation is that large amount of ligand molecules may crosslink with each other on the surface of QDs, resulting in

Fig. 4. Effects of pH (left), reaction time (middle) and reaction temperature (right) on fluorescence emission spectra of prepared CdTe QDs.

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Fig. 5. Fluorescence emission spectra and emission intensity (insert) of CdTe QDs synthesized with different molar ratio of ligand/Cd (left) at the molar ratio of TGA to GSH of 1:1 and fluorescence intensity of CdTe QDs with different molar ratio of TGA to GSH (right) at the molar ratio of ligand/Cd of 1.2:1.

more surface defects. Another possible reason likely ascribes the probable crosslinking of the ligand molecules between different QDs, which leads to their aggregation. This is visible in the spectral broadening with increasing the amount of stabilizer. Decrease in the emission intensity may be due to the quenching effect with increasing aggregation of the molecules as seen in both left and right panel. The effect of the ratio of TGA and GSH on the intensity of prepared CdTe QDs was also investigated. As shown in Fig. 5(right), compared with either TGA or GSH alone, the TGA–GSH co-capped QDs showed stronger fluorescence intensity, with the optimal TGA/GSH ratio at 1:1. This may be attributed to the structural complementation of TGA and GSH molecules. The relative small TGA molecule with strong bond of S H to Cd can effectively coat QDs surface. On the other hand, the longer GSH molecular chains can bend to arc-shape to adequately cap the surface, even though their S H can from relative weak bond with Cd. The synchronous co-capping of TGA and GSH can provide both effective binding and sufficient capping, leading to the enhancement of fluorescence intensity of the prepared QDs. 3.4. Advantages of the GSH–TGA co-capped CdTe QDs The optical stability of as prepared GSH–TGA co-modified CdTe QDs (GSH–TGA-QDs) was compared with that of GSH or TGA solely modified CdTe QDs (GSH-QDs and TGA-QDs, respectively). As shown in Fig. 6(left), under ultraviolet light, TGA-QDs showed good optical stability in 3 h. The reason for this could be that the S2− generated from TGA’s degradation combined with Cd2+ to form

wideband CdS coating layer capped on the surface of CdTe QDs. The decrease of fluorescence intensity is most likely due to the new surface defects caused by the increase of irradiation time. In contrast, the fluorescent intensity of GSH-QDs decreased rapidly, reducing 20% in 5 min and 60% in 60 min. This can be explained by the fact that the GSH–Cd interaction is much weaker than TGA–Cd. The GSH’s degradation is too rapid to form CdS shell, resulting in the separation of GSH from the surface of CdTe QDS and leading to lot of surface defects. Even though GSH-QDs possess excellent biocompatibility characteristics, the unsatisfactory fluorescence instability is the limiting factor for them to be used in bio-application. Fortunately, we have successfully prepared the GSH–TGA-QDs with obviously improved optical stability. With fluorescence intensity decreased only by 21% in 1 h, these QDs can be used without concerns on the influence on bio-applications. At same reaction condition, the emission wave length of TGAQDs, GSH-QDs and GSH–TGA-QDs obtained with reaction time of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 h are shown in Fig. 6(right). With short reaction time, the maximum emission positions of the three QDs are almost identical. However, the difference in emission wavelength appeared and increased with the prolonging of reaction period. As mentioned previously, for the small TGA molecules, the strong interaction between SH and Cd resulted in stronger binding force to the CdTe QDs’ surface, which retarded the growth of crystals to larger size. Whereas for the long-chain GSH molecules, the weak interaction between SH and Cd make the binding force to the CdTe QDs’ surface weaker, leading to rapid growth speed. In addition, due to the interactions between the stabilizers in different

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Fig. 6. Variation in fluorescence intensity under ultraviolet light (left) and time-dependent variation in emission peak position (right) of three QDs.

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Fig. 7. Mechanism for the immunolabeling of colorectal cancer cells using CdTe QDs.

QDs, the aggregation of the QDs with their prolonged exposure to irradiation may also be a reason for the shift in emission maximum position and decrease in emission intensity. The emission wavelength of GSH–TGA-QDs is located between TGA-QDs and SGH-QDs. In summary, compared to GSH or TGA solely capped CdTe QDs, the GSH–TGA co-capped CdTe QDs offered several advantages: the fluorescent intensity was enhanced by about one-fold, the emission wavelength range was expanded to 511–641 nm, and the optical stability was improved significantly compared with GSH-QDs. Traditionally, the synthesis of QDs with core/shell structure was used for the enhancement of fluorescence intensity and optical stability. In our study, the two types of complementary ligands, TGA and GSH, were used for the one-step preparation of CdTe QDs, providing a new approach for preparing high quality QDs.

3.5. Immunolabeling and imaging of colorectal cancer cells Monoclonal antibody ND-1 (MoAb ND-1) is the anti-human colon carcinoma antibody prepared by using colon carcinoma cell CCL187 as immunogen. MoAb ND-1 specifically recognize LEA (large external antigen, LEA), a tumor related antigen overexpressed on colon carcinoma cells with higher selectivity on well differentiated colon carcinoma. Here, we prepared the specific fluorescent immuno probe, GSH–TGA-QDs-ND-1, by using NHS and EDC as activator to induce the coupling reaction. EDC can react with carboxyl groups on the surface of GSH–TGA-QDs at moderate conditions to form a hot activity ester, which is able to react with the amino groups on GSH–TGA-QDs. Unfortunately, as a midbody, this hot activity ester is easily hydrolyzed, the addition of NHS could enhance the reaction yield by forming an esters-like mid-body which is not readily hydrolyzed and able to react with the amino groups to form amido bond. Subsequently, the activated carboxyl groups on the surface of GSH–TGA-QDs react with the amino groups of antibody by covalently coupling to generate the LEA specific immune-probe GSH–TGA-QDs-ND-1. The tumor related antigen LEA is overexpressed on the surface of well differentiated colon carcinoma cell, but is not expressed or very lowly expressed in normal tissues, non-colon carcinoma or poorly differentiated tissues. The mechanism for immunolabeling of colorectal cancer cells using the as-prepared CdTe QDs is shown in Fig. 7. The colon carcinoma cell line CCL187 was labeled through specific immuno-reaction of probe GSH–TGA-QDs-ND-1 with LEA. As shown in Fig. 8(a), colon carcinoma cell CCL187 alone almost does not show any green fluorescence (at 488 nm) under UV radiation. However, as can be seen in Fig. 8(c), after co-incubated with GSH–TGA-QDs-ND-1 probes, the living colon carcinoma cell CCL187 were successfully labeled and imaged. These results suggest that through the specific interaction of antigen LEA and antibody ND-1, the probes can be successfully presented on the surface of cells membrane, some of them even might enter into the cells making the intracellular fluorescence appeared. In a control

Fig. 8. Fluorescent images of colorectal cancer cells: (a) cells alone incubated for 1 h; (b) cells incubated with QDs for 1 h; (c) cells incubated with QDs-ND-1 for 1 h; the rows from left to right are the images in bright field, dark fields, and overlays respectively.

experiment, where the nanoparticles without antibody conjugation are incubated with the cells, the cells showed only minimal green light as seen in Fig. 8(b). This observation supports the theory that the interaction between the carboxyl groups on GSH–TGAQDs and the antigens on the cell membranes will not influence the immuno-labeling of the cells. 4. Conclusion In summary, the CdTe QDs with good biological compatibility and high fluorescent intensity were synthesized hydrothermally by using GSH and TGA as co-capped ligands. The synthesis conditions such as reaction time, temperature, pH value, ligands concentration and the molar ratio of GSH:TGA were carefully optimized. The experimental results of optical properties and inner structures of as-prepared CdTe QDs showed that both fluorescence intensity and optical stability of the GSH–TGA co-capped CdTe QDs were significantly improved. Finally, the as-prepared QDs were conjugated with monoclonal antibody ND-1. By the specific reaction between antibody ND-1 and antigen LEA, GSH–TGA-QDs-ND-1 probes were successfully used to label colorectal cancer cells CCL187. Control experimental results indicated that the labeling of colorectal cancer cells by QDs-ND-1 was specific. Compared with other methods, the direct labeling procedure was relatively simpler and more

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