КОЛЛОИДНЫЙ ЖУРНАЛ, 2010, том 72, № 5, с. 701-706
УДК 541.18
ONE-POT SYNTHESIS OF STRONGLY LUMINESENCING CDTE QUANTUM DOTS AND THEIR CONJUGATION WITH MOUSE ANTIBODY
TO ALPHA-FETOPROTEIN
© 2010 г. Jing Li*, Xingping Zhou*1, Siyu Ni*, Xiaqin Wang**1
* College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, P.R. China ** College of Material Science and Engineering, Donghua University, Shanghai 201620, P.R. China Поступила в редакцию 03.11.2009 г.
In this paper, strongly luminescent CdTe quantum dots (QDs) were synthesized in aqueous solution by a facile one-pot method. The CdTe QDs were synthesized in a weakly acidic or neutral buffer solution composed of sodium borate (Na2B4O7) and sodium citrate (C6H5Na3O7). The pH of buffer solution and the ratio of the precursors were systematically optimized; the high-quality CdTe QDs with progressively increasing fluorescence during 60 days storage were obtained. As-prepared QDs can be conjugated with a mouse antibody al-pha-fetoprotein via the reaction mediated by Ж-hydroxysuccinimide and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride. The conjugate showed a red shift of 9 nm for the emission position.
1. INTRODUCTION
Recently, aqueous solutions of quantum dots (QDs) have drawn much attention because of their excellent spectral properties and high photoluminescence quantum efficiency (QE) [1, 2]. When compared to organic synthesis, the process in aqueous solution has several advantages such as simplicity, reproducibility, and low tox-icity [3]. However, for the majority of aqueous synthesis methods, H2Te (the product of chemical decomposition ofAl2Te3 powder) [4, 5] or NaHTe (obtained via reaction of Te powder with NaBH4) [6—10] are utilized as tellurium precursors, which require long-term synthetic procedures. On the other hand, the photoluminescence of CdTe QDs synthesized in the aqueous phase is generally low (<20%) [11, 12], and the particles are rather unstable [11]. The development of reliable and simple method for the formation of luminescent QDs in aqueous solution remains a challenge in this field. The selection of an appropriate thiol stabilizers and optimization of the reaction conditions allow the significant enhancement of stability and fluorescent properties of CdTe QDs [13, 14].
The traditional immunofluorescence technology utilizes organic dyes (Rhodamine, fluorescein isothiocyan-ate and so on) to label specific antibody to dye cell, bacterium, virus, tissue section and liquid substance. However, these organic dyes are generally vulnerable to physiological environment and are quickly pho-tobleached under common conditions [15]. In contrast, QDs exhibit superior fluorescent properties, small size, emission tunability, photostability and long photoluminescence (PL) decay time [16], and, therefore, these ma-
1 Corresponding authors, xqwang@dhu.edu.cn
E-mails: xpzhou@dhu.edu.cn/
terials can be applied to immunoanalysis and immunodetection [17—19], optoelectronic devices [20], photovoltaic devices [21] and biological fluorescence labeling [22]. To the best of our knowledge, the standard requirement for all these applications is a weakly acidic or neutral environment. However, CdTe QDs are usually synthesized in alkaline aqueous solution [6—10] for obtaining a relatively strong luminescence and such QDs cannot be used in biological studies. Furthermore, the electrostatic attraction, which commonly underlies the application of QDs, is strongly influenced by the pH of the media.
In this paper, we systematically studied a facile one-pot synthetic approach, and demonstrated that strongly luminescencing CdTe QDs could be synthesized in a weakly acidic (pH 6.0—7.0) solution by using sodium tellurite (Na2TeO3) as a tellurium source and thioglycolic acid (TGA) as a capping ligand. This makes the as-prepared QDs suitable for biological or medical application. The fluorescence performance of CdTe QDs was drastically improved after 60 days storage through optimizing the preparation conditions. The TGA-capped CdTe QDs were used for direct conjugation with alpha-fetoprotein (anti-AFP) by covalent binding via ^-hydroxysuccinim-ide (NHS) and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). When compared to electrostatic interactions, the covalent binding gives more stable product with low sensitivity to physiological environment.
2. EXPERIMENTAL
2.1. Reagents and Instruments
The reagents used in the experiments were of analytical grade and used without further purification.
Absorbance, a.u 0.70
0.60 L 3
0.50 0.40 0.30 0.20 0.10 0
2' 3
PL Intensity, a.u.
70
60 50 40 30 20 10 0
400 450
500 550 600 Wavelength, nm
650 700
10
Fig. 1. (1-3) UV-Vis and (1—3') PL spectra of CdTe QDs prepared at reaction time of (1, 1') 45, (2, 2') 75, and (3, 3') 105 min.
Anti-AFP was purchased from Shanghai Linc-Bio Science Co. LTD, TGA (97%) was from A Johnson Matthey Company, and EDC was from J&K Chemical Ltd. Dialysis membranes of 10—12 kD cut off range were from Spectra/Pordialysis, USA. Other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. Milli-Q grade water (18.2 Mfi) was used for the preparation of aqueous solutions.
Photoluminescence of CdTe QDs was measured with a Hitachi F-4500 spectrofluorometer equipped with Xe lamp. UV spectrometry of CdTe QDs was conducted with an ultraviolet—visible spectrophotometer, JASCO V-530, with the light path of 1 cm. TEM images were obtained using a Hitachi H-800 electron microscope operating at 200 kV Power X-ray diffraction (XRD) measurements were performed on D/max-2550PC X-ray diffrac-tometer using Cu Ka radiation. The quantum efficiency was determined by comparison with Rhodamine 6G in aqueous solution.
2.2. Synthesis of TGA-Capped CdTe QDs
CdTe QDs were synthesized according to the procedure described by Ying [3]. Buffer solution of 15 mM Na2B4O7 and 15 mM C6H5Na3O7 with the pH adjusted to the desired value with 1.0 M HCl was injected into a two-neck flask under vacuum and then saturated with nitrogen. The precursor solutions of CdCl2 (0.1 M), Na2TeO3 (0.4 M) and TGA (1.2 M) were successively added to the flask. After vigorous stirring for 5 min, 20 mg of NaBH4 powder was added rapidly to the precursor solution. When the solution began to turn to yellow, the flask was attached to a condenser and refluxed at 95—100°C under nitrogen atmosphere. A series of CdTe QDs with different sizes were obtained by heating the products in boiling water bath for various periods.
2.3. Conjugation with Mouse Anti-AFP Antibody
500 ^l TGA-Capped CdTe QDs was initially activated using NHS (0.05 M), and then kept at 37°C for 10 min. Then, 1 mg ofanti-AFP and EDC (0.05 M) was added to the mixed solution and equilibrated at 37°C. Finally, a portion of glycine (1.0 M) was added for the reaction completion. The sample was later dialyzed using Spectra/Pordialysis bag (PBS) with a molecular weight cut off limit of 10-12 kD for 12 h; PBS containing 0.02% of TGA was changed 4 times to remove residual NHS, EDC and CdTe.
3. RESULTS AND DISCUSSION
3.1. Synthesis of TGA-capped CdTe QDs with Different Sizes
When the clear precursor solution containing CdCl2, Na2TeO3, TGA, and NaBH4 was heated in boiling water bath, its color gradually changed to green with time. The heating produced a lot of bubbles released from the solution. No luminescence was, however, observed in this solution because of the very small size of initially formed CdTe QDs. After heating of several minutes the weak luminescence became detectable. The luminescence of the precursor solution increases with the reaction time, and this increase is accompanied with changes of solution color. Due to the quantum confinement effect, which depends on the QD size, green, yellow, and orange TGA-capped CdTe QDs can be synthesized by varying the reaction time.
When the reaction time is increased, the characteristic bands in the absorption spectra and the PL emission spectra of CdTe QDs are shifted to a longer wavelength that corresponds to the increasing size of QDs. This shift is related to the decrease of the effective band gap of QDs with increasing size, when its value is close to that ofBohr radius. This results in a series of discrete energy levels in energy bands. Accordingly, the absorption spectra shift to shorter wavelengths as the size of QDs decreases. Figure 1 shows typical UV-Vis spectra and PL intensities of different samples of CdTe QDs. These samples were synthesized in the buffer solution at pH 6.24. During the 105 min reaction, the absorption peak of CdTe QDs was shifted from 473 to 528 nm, and the PL emission peak of CdTe QDs was shifted from 515 to 555 nm, indicating the quantum confinement effect.
3.2. Optimization of the Parameters for CdTe QDs Synthesis
Effects of the ratio of the precursors and pH value of the solution on synthesis of CdTe QDs and that of the storage time were systematically investigated in the experiments.
It is well known that the pH value is one of the most important factors, which often affects the optical properties of CdTe QDs prepared in aqueous solution. Figure 2 shows that stable CdTe QDs can be obtained in the pH
PL Intensity, a.u.
60
50 40 30 20 10 0
400 450 500 550 600 650 700
Wavelength, nm
Fig. 2. PL spectra of CdTe QDs prepared at pH values of (1) 6.24, (2) 6.78, (3) 6.87, (4) 7.27, and (5) 8.24.
Absorbance, a.u. 0.40
0.35 0.30 0.25 0.20 0.15 0.10 0.05 0
0.05 0.10
3
3 '
2 2 '
1
t; ' 1 ■
У 7. ■м 1 ' ■■
PL Intensity, a.u.
60 50 40 30 20 10 0
400 450 500 550 600 650 700 Wavelength, nm
Fig. 3. (1-3) UV-Vis and (13') PL spectra of CdTe QDs prepared at [Cd2+]/[TeO32-] ratio of (1, 1') 5.0, (2, 2') 6.0, and (3, 3') 7.0.
10
range of 6.0—7.0. The CdTe QDs with the strongest fluorescence intensity were formed at pH 6.24. Therefore, pH 6.24 was selected for the further investigations. Since the dissociation ofthe cadmium thiol complexes
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.