ZnS QDs

Journal of Luminescence 132 (2012) 2484–2488

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Determination of rifampicin based on fluorescence quenching of GSH capped CdTe/ZnS QDs Zhengqing Liu, Pengfei Yin, Huiping Gong, Pingping Li, Xiaodan Wang, Youqiu He n School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 December 2011 Received in revised form 22 March 2012 Accepted 27 March 2012 Available online 12 April 2012

Aqueous glutathione (GSH)-capped CdTe/ZnS QDs with the diameter of 3–4 nm were synthesized. The fluorescence of CdTe/ZnS QDs at 577 nm was quenched in the presence of rifampicin (Rfp), with excitation wavelength at 350 nm. The mechanism of the interaction of CdTe/ZnS QDs with Rfp was investigated. Under the optimal conditions, the calibration plot of ln(F0/F) was linear in the range 0.83– 56 mg mL  1 with concentration of Rfp, and the detection limit was 0.25 mg mL  1. The proposed method was successfully applied to the determination of Rfp in its commercial capsules, and satisfactory results were obtained. The recovery of the method was in the range 98.6–103.2%. & 2012 Elsevier B.V. All rights reserved.

Keywords: Quantum dots Rifampicin Fluorescence quenching Determination

1. Introduction Quantum dots (QDs) are a brand new class of fluorescent nanoprobes, the semiconductor particles have all three dimensions confined to the 1–10 nm length scale [1]. As a result of quantum confinement, they have unique optical and electronic properties such as tunable particle size, high quantum yield, broad excitation spectrum, narrow and tunable emission spectrum, high photobleaching threshold and long-term photostability [2–4]. QDs have attracted considerable attention as novel luminescence probes in recent years [5–9], such as the determination of paeonol [10], roxithromycin [11], ascorbic acid [12], sulfadiazine [13] in analytical chemistry. They believe that it is possible to develop sensitive and simple sensors by using semiconductor QDs. These studies reveal that the interactions between some substances and the QDs would change their photophysical properties. Rifampicin (Rfp) is an important semisynthetic anti-tuberculosis drug derived from Streptomyces mediterranei. It is a bactericidal agent, with sterilizing activity against Mycobacterium tuberculosis. It binds to the subunit of the DNA-dependent RNA polymerase and inhibits the initiation of transcription [14]. Many methods have been reported for the determination of Rfp, such as HPLC [15,16], Chromatography [17], and Real-Time PCR [18]. Although these methods are generally very sensitive and accurate, they tend to be

n

Corresponding author. Tel.: þ86 23 68367475; fax: þ86 23 68254000. E-mail address: [email protected] (Y. He).

0022-2313/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jlumin.2012.03.072

cost-effective, technically complex, time-consuming, and do not allow high throughput analysis. So the development of a rapid and sensitive method for the quantitative analysis of Rfp is required. To our knowledge, the use of CdTe/ZnS QDs as fluorescence probe for the quantitative determination of Rfp has not been reported so far. In this article, we investigated the interaction between CdTe/ZnS QDs and Rfp. It was found that the fluorescence intensity of CdTe/ZnS QDs was quenched at 577 nm in the presence of Rfp, and the quenched intensity was proportional to the concentration of Rfp in the range 0.83–56 mg mL  1 with a detection limit of 0.25 mg mL  1. The method has been applied to determination of Rfp in its commercial samples and satisfactory results were obtained. The mechanism of the proposed reaction was also discussed.

2. Experimental 2.1. Apparatus A Hitachi F-2500 spectrofluorophotometer (Hitachi Company, Japan) was used to record the fluorescence spectra. A UV-8500 spectrophotometer (Tianmei Corporation, Shanghai, China) was applied to record the absorption spectra. Hitachi-600 transmission electron microscopy (TEM, Hitachi Company, Japan) was adopted to examine the appearance and size of nanoparticles. A PHS-3C pH meter (Leici, Shanghai, China) was utilized to measure the pH values of the aqueous solutions.

2485

0.25

3500

The main chemical reagents used in the present study are CdCl2  2.5H2O (Shanghai Chemicals Reagent Co., Shanghai, China), Te powder (Sinopharm Chemical Reagent Co., Shanghai, China), Thioglycolic acid (TGA, Sinopharm Chemical Reagent Co., Shanghai, China), glutathione (GSH, Aladdin Reagent Co., Shanghai, China), NaBH4 (Tianjin Huanwei Fine Chemical Co., Tianjin, China), rifampin (Rfp, Aladdin Reagent Co., Shanghai, China), rifampin capsules (ChengDu Jinhua Pharmaceutical Co., Ltd., Chengdu, China); Tris–HCl buffer solutions with different pH values were prepared according to suitable proportions. All reagents used were of analytical grade without further purification. Water used throughout was doubly deionized.

0.20

2800

0.15

2100

2.3. Methods 2.3.1. Synthesis of GSH–CdTe/ZnS QDs CdTe/ZnS QDs stabilized by GSH were synthesized according to the previously described method [19]. For a typical reaction, Te powder (0.0255 g) was placed in a 50 mL three-necked flask. Excessive sodium borohydride was added under magnetic stirring. Finally, the colorless solution of NaHTe was prepared. Then CdCl2  2.5H2O (0.1826 g) was dissolved in 150 mL deionized water, 88 mL TGA stabilizer was added under vigorous stirring and the pH was adjusted to 11–11.5 by dropwise addition of 1 mol L  1 NaOH solution. Then H2SO4 (0.5 mol L  1) was introduced to NaHTe to produce H2Te gas, which passed through the cadmium precursor with a slow N2 flow for 30 min. CdTe precursors were formed at this stage. The molar ratio Cd2 þ / TGA/HTe  was fixed at 1:1.5:0.25. The resulting mixture was subjected to reflux at 369 K for 0.5 h under open-air condition with condenser. Then 1 mL solution (pH¼8) containing 0.0136 g ZnCl2 and 0.1229 g GSH was added to the as-prepared CdTe solution. The solution was heated to 373 K under open-air conditions and refluxed for another 2 h. Eventually, the salmon pink CdTe/ZnS QDs quantum dots were obtained. 2.3.2. Analytical procedure At this stage, 1.4 mL above prepared CdTe/ZnS QDs, an appropriate amount of Tris–HCl solution, and Rfp were added into a 10 mL volumetric flask, then diluted with deionized water to the mark, and mixed thoroughly by gentle shaking. After incubation for 10 min, the spectra of solution were examined. For the pharmaceutical analysis, one capsule of rifampin was reduced to homogeneous fine powder. Added five drops of 1 mol L  1 NaOH to facilitate dissolving the rifampicin. After sonicated for 10 min, the solution was transferred into a 100 mL calibrated flask and made up to the volume with distilled water.

Abs

2.2. Reagents

0.10

b

a

1400

FL intensity

Z. Liu et al. / Journal of Luminescence 132 (2012) 2484–2488

700

0.05

400

500

600

700

/nm Fig. 1. (a) Absorption spectra and (b) fluorescence spectra (excited at 350 nm) of CdTe/ZnS QDs.

Fig. 2. TEM image of aqueous CdTe/ZnS QDs.

The particle size of CdTe/ZnS QDs can be estimated about 3.4 nm from the absorption maximum of 546 nm (Fig. 1) by the following equation [20], which is in good agreement with the TEM observation results. 3

2

D ¼ ð9:8127  107 Þl ð1:7147  103 Þl þ ð1:0064Þl194:84

ð1Þ

3.2. Fluorescence quenching of CdTe/ZnS QDs by Rfp The fluorescence spectra of CdTe/ZnS QDs were recorded in the absence and presence of Rfp and shown in Fig. 3. CdTe/ZnS QDs exhibited strong fluorescence and the observed fluorescence band centered at 577 nm (excitation 350 nm). When Rfp was added to the CdTe/ZnS QDs, the fluorescence of CdTe/ZnS QDs was quenched sharply, based on this, the possibility of developing a sensitive method for Rfp has been evaluated.

3. Results and discussion 3.3. Optimization of the reactions 3.1. Spectral characterization of CdTe/ZnS QDs The fluorescence and absorption spectra of the CdTe/ZnS QDs were obtained and shown in Fig. 1. The fact that the emission maximum was close to its absorption onset indicated the emission arose from direct recombination of charge carriers between conduction and valence bands. The as-prepared CdTe/ZnS QDs exhibited narrow fluorescence bandwidth as the consequence of homogeneous size distribution and uniform crystallinity, which was confirmed by TEM. TEM images of aqueous CdTe/ZnS QDs are shown in Fig. 2, the particles are monodisperse in shape and the sizes are around 3–4 nm.

3.3.1. Effect of the acidity The influence of acidity on the fluorescence intensity of aqueous CdTe/ZnS QDs–Rfp solution system was investigated and shown in Fig. 4. In this paper, Tris–HCl was used to control the acidity of analytical system. The results showed that the maximum value of F0–F was obtained when pH was 7.6. In order to obtain a lower detection limit, so pH 7.6 was chosen to be the optimal reaction pH in this experiment. 3.3.2. Effect of aqueous CdTe/ZnS QDs concentration The effect of CdTe/ZnS QDs concentration on the fluorescence intensity of CdTe/ZnS QDs-Rfp solution system was studied by

2486

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3500

900

1.6 1.4 1.2 ln (F /F)

2800

0.8 0.6

700

0.4 0.2

2100

0.0 -0.2 0

10

20 30 40 c (µg·mL )

50

60

F0-F

FL intensity

1

800

1.0

600

1400 500

8 700

400 0.6

450

500

550

600 650 /nm

700

750

0.8

1.0

1.2 1.4 c10−4 (mol·L−1)

800

Fig. 3. Fluorescence spectra of CdTe/ZnS QDs-Rfp system cQDs: 2.8  10–4 mol L  1 , cRfp (1–8): 0, 8, 16, 24, 32, 40, 48 and 56 mg mL  1.

1.6

1.8

Fig. 5. Effect of the concentration of aqueous CdTe/ZnS QDs. Fluorescence intensity of the solution system in the presence (F) and the absence(F0) of Rfp (cRfp: 8 mg mL  1), pH¼ 7.6.

2400

650 2350

FL intensity

600

F0-F

550 500

2300

2250

450 2200 0

400

7.0

7.2

7.4

7.6

7.8 pH

8.0

8.2

8.4

8.6

Fig. 4. Effect of pH on fluorescence intensity of the solution system in the presence (F)and the absence (F0) of Rfp (cRfp: 8 mg mL  1, cQDs: 2.0  10  4 mol L  1).

keeping the Rfp concentration and the pH constant while changing the CdTe/ZnS QDs concentration. As shown in Fig. 5, the CdTe/ZnS QDs concentration strongly influenced the fluorescence intensity of the solution system and the optimum concentration of CdTe/ZnS QDs was 2.8  10  4 mol L  1. The value of F0 –F decreased when the addition amount of CdTe/ZnS QDs went beyond this concentration. Accordingly, the CdTe/ZnS QDs concentration of 2.8  10  4 mol L  1 was chosen to be the optimum concentration for the solution system.

3.3.3. Effect of incubation time The influence of incubation time on the fluorescence intensity was investigated at different time scales at room temperature and the results are shown in Fig. 6. It was found that the mixture showed a rapid decrease in fluorescence spectrum intensity after the Rfp was added. The reaction was completed within 10 min at room temperature, indicating that certain time was needed to complete the interaction, and the fluorescence intensity remained stable for at least 1.5 h. Therefore, the experiments were carried out after 10 min and the time scale of 10 min was also adopted in the following experiments.

20

40

60 t (min)

80

100

120

Fig. 6. Effect of incubation time (cRfp: 8 mg mL  1, cQDs: 2.8  10  4 mol L  1).

3.4. Possible interaction mechanism It is known that fluorescence quenching can be classified into two categories, static and dynamic quenching, they can be distinguished by their differing dependence on temperature [21]. The quenching rate constants increase with increasing temperature for dynamic quenching, but the reverse effect is observed in case of static quenching [22]. The fluorescence quenching mechanism can be analyzed quantitatively at different temperatures (299, 304 and 309 K) with the Stern–Volmer equation [23] F 0 =F ¼ 1þ K q t0 ½Q  ¼ 1 þ kSV ½Q 

ð2Þ

where F and F0 are the fluorescence intensity of CdTe/ZnS QDs in the presence and absence of quencher, respectively, Kq is the quenching constant, kSV is the Stern–Volmer quenching constant, t0 is the fluorescence lifetime in the absence of quencher, and [Q] is the concentration of quencher. Fig. 7 shows the Stern–Volmer plots of F0/F versus [Q] at three different temperatures. kSV of the CdTe/ZnS QDs–Rfp solution system at three different temperatures were calculated according to Eq. (2) and listed in Table 1. It can be seen that the quenching constants increased with rise of temperature, which indicates that the quenching type of CdTe/ZnS QDs–Rfp solution system is dynamic quenching. To explore the mechanism of reaction, we explored transfer of electron from CdTe/ZnS QDs to Rfp. It is well known that upon

Z. Liu et al. / Journal of Luminescence 132 (2012) 2484–2488

Rfp in solution are shown in Fig. 8. In the spectrum of pure QDs, there is strong absorption in the UV at wavelengths o400 nm, whereas the absorption in the visible is relatively weak. In the spectrum of pure Rfp, there are two strong absorption peaks at 333 nm and 472 nm. No indication of a spectral shift is observed as Rfp is added. For the mixture of QDs and Rfp, the absorption spectrum is simply a superposition of the QDs and Rfp absorption spectra. Fig. 8 clearly shows the absorption spectrum of the mixture is a linear combination of the spectra of each component, implying dynamic quenching.

5.0 299K 304K 309K

4.5 4.0 3.5

F0/F

2487

3.0 2.5 2.0 1.5

3.5. Calibration curves and sensitivity

1.0 2

3

4 5 [Q] (10–5 mol·L–1)

6

7

Fig. 7. Stern–Volmer plots for the CdTe/ZnS QDs–Rfp solution system at three different temperatures (cQDs: 2.8  10  4 mol L  1, pH ¼7.6).

Table 1 Parameters of Stern–Volmer plots of CdTe/ZnS QDs–Rfp solution system. Temperature (K)

Sterm–Volmer linear equation

kSV (mol L–1)

Correlation coefficient

299

F 0 =F ¼ 0:432 þ 5:27  104 ½Q 

5.27  104

0.9966

304

F 0 =F ¼ 0:482 þ 5:49  104 ½Q 

4

5.49  10

0.9986

309

F 0 =F ¼ 0:538 þ 5:73  104 ½Q 

5.73  104

0.9987

Under the optimal conditions, the fluorescence spectra of aqueous CdTe/ZnS QDs with different concentrations of Rfp were recorded, the results are shown in Fig. 3. Within the concentration range 0.83–56 mg mL  1, quenching aqueous CdTe/ZnS QDs by Rfp satisfied the following equation: ln(F0/F)¼0.02803cþ0.03282, F0 and F are the fluorescence intensities of the CdTe/ZnS QDs without and at a given Rfp concentration, the correlation coefficient is 0.9986. The limit of detection (3d/ K  1) is 0.25 mg mL  1.

3.6. Interference of co-existing foreign substances In order to investigate the possibility of practical application of the procedure, the interference from some familiar foreign ions and excipients (which were often contained in commercial capsules, such as glycerol, triethanolamine and phthalic acid) were tested under the optimum conditions. From the results displayed in Table 2, we can see that most of the common metal ions, glucide, amino acids and excipients could be allowed at high concentration, whereas Fe3 þ and Cu2 þ could be allowed at lower concentration levels without significant interference.

3.7. Analytical application

excitation of CdTe/ZnS QDs, it would result in the promotion of electron from its valence band to the conduction band. This results in the formation of a positively charged hole in its valence band and a free electron in the conduction band of QDs. In the absence of Rfp, the recombination of the electron and hole results the fluorescence. While introducing Rfp (which serves as efficient electron acceptor for the conduction band electron from the CdTe/ ZnS QDs [24,25]) to the solution of QDs prevented the electron– hole recombination at the interfaces of CdTe/ZnS QDs (Scheme 1), which caused the fluorescence quenching [26]. In dynamic quenching, charge transfer occurs and the fluorescence is quenched when the electron acceptor collides with the excited fluorophore. Because the collision between the quencher and the fluorophore affects only the excited state of the fluorophore, no changes in the absorption spectrum are expected. On the contrary, the formation of ground-state complex in static quenching will perturb the absorption spectra of the fluorophore [27]. Thus, by careful examination of the absorption spectrum, one can attempt to distinguish static and dynamic quenching. The absorption spectra of QDs in solution, of Rfp in solution, and QDs–

1.2 1.0

c

0.8

Abs

Scheme 1. Mechanism of fluorescence quenching process.

The practical feasibility of the fluorescent probe was applied to determine Rfp in commercial capsules. The results are shown in Table 3 and recoveries of the spiked samples were generally satisfactory. The accuracy of the developed method was evaluated by analyzing the samples with the reference (HPLC method [16] for the assay of Rfp was adopted) procedures. The results obtained by the developed system are in good agreement with those by the HPLC method.

0.6 0.4

b 0.2

a 300

400

500

600

/nm Fig. 8. UV–vis absorption spectra of (a) CdTe/ZnS QDs, (b) Rfp, and (c) the mixture (CdTe/ZnS QDs and Rfp) (cRfp: 8 mg mL  1, cQDs: 2.8  10  4 mol L  1).

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Z. Liu et al. / Journal of Luminescence 132 (2012) 2484–2488

Table 2 Effects of coexistent substances of CdTe/ZnS QDs–Rfp solution system. (cRfp: 8 mg mL  1). Coexisting substances

Coexisting concentration (mg mL  1)

Relative error (%)

Coexisting substances

Coexisting concentration (mg mL  1)

Relative error (%)

l-cysteine l-tryptophan l-glutamic acid Phthalic acid Glycerol Triethanolamine HSA Pepsase Glucose

60 60 60 15 800 1000 50 40 840

þ2.2 þ2.7 þ3.2 þ3.1 þ1.4 þ2.3 þ3.8 þ2.3 þ 0.6

Sucrose K þ (SO42  ) Cu2 þ (SO42  ) Ba2 þ (Cl  ) Zn2 þ (Cl  ) Ca2 þ (NO3  ) Mg2 þ (SO42  ) Fe3 þ (SO42  ) Na þ (Cl–)

1000 400 1.0 10 14 26 80 1.5 400

þ 1.6 þ0.3  2.3 þ 5.4 þ 4.7 þ 1.9 þ 3.3  3.1 þ0.5

References

Table 3 Result for the determination of Rfp in commercial capsules. Rfp capsules samplesa

Reference Proposed R.S.D. (%) procedures (mg)b procedures (mg) (n¼ 5)

Average recovery (%)

1 2 3

149.7 149.5 150.2

103.2 99.5 98.6

a b

149.1 149.3 149.4

3.5 2.3 3.1

Rfp capsules is 150 mg. HPLC method.

4. Conclusion A novel and convenient method for Rfp analysis has been established based on the fluorescence quenching of aqueous CdTe/ZnS QDs. The preparation of CdTe/ZnS QDs is simple and inexpensive. Under the optimum conditions, a good linear relationship between fluorescence intensity of the system and the concentration of Rfp in the range 0.83–56 mg mL  1 could be achieved, and the limit of detection is 0.25 mg mL  1. Rfp could be sensitively detected with this simple setup, even though the sensitivity of the proposed method is not higher than that of HPLC method. When the proposed method was applied in the determination of Rfp in capsules, the results are in excellent agreement with the claimed value. The assay is characterized by simplicity, rapidity, and high sensitivity.

Acknowledgments The work is supported by the National Natural Science Foundation of China (No. 20875078), Chongqing Municipal Key Laboratory on Luminescence and Real-Time Analysis, and Southwest University (No. CSTC 2006CA8006).

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