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Simple and sensitive detection method for diprophylline using glutathione-capped CdTe quantum dots as fluorescence probes
Journal of Luminescence 145 (2014) 575–581
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Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Simple and sensitive detection method for diprophylline using glutathione-capped CdTe quantum dots as fluorescence probes Suyan Ying a, Shumin Cui a, Weiping Wang a,n, Jiuju Feng a, Jianrong Chen b a b
College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, China College of Geography and Environmental Science, Zhejiang Normal University, Jinhua 321004, China
art ic l e i nf o
a b s t r a c t
Article history: Received 18 October 2012 Received in revised form 4 August 2013 Accepted 14 August 2013 Available online 24 August 2013
A simple and sensitive method for detecting diprophylline (DPP) was developed based on the fluorescence quenching of glutathione-capped CdTe quantum dots (GSH–CdTe QDs) by using diprophylline in a KH2PO4–Na2HPO4 medium. Parameters affecting the quenching efficiency, including types and pH of buffer solutions as well as temperature, reaction time, adding sequence, and interfering substances, were investigated and optimized. In optimum conditions, the calibration plot of the quenched fluorescence intensity F 0 =F with a DPP concentration range of 1.67 10–6 mol L 1 to 1.33 10–5 mol L 1 –7 was linear. The detection limit (with signal to noise ratio of 3) for DPP was 2.24 10 mol L 1. The proposed method was successfully applied for detecting DPP in human serum. The recovery of the method was in the range of 87.41% to 117.94%. Finally, the possible quenching mechanism of GSH–CdTe QDs and DPP was also discussed. & 2013 Elsevier B.V. All rights reserved.
Keywords: Glutathione-capped CdTe quantum dots Diprophylline Fluorescence quenching
1. Introduction Diprophylline (DPP) is a methylxanthine drug that can directly relax the smooth bronchi muscles and pulmonary blood vessels. It is usually employed for the symptomatic relief or clinical prevention of bronchial asthma and chronic obstructive pulmonary disease [1]. Compared with theophylline, DPP has an enhanced water solubility and fewer adverse reactions. The main metabolites of DPP in blood are its prototype and theophylline derivatives. After 24 h of medication, 83% of DPP is excreted through urine. However, the transport and metabolic process of DPP in vivo remain unclear. Wang et al. [1] investigated the binding characteristic of DPP with lysozyme via spectroscopic methods. Until now, the detection and quantification of DPP in biological fluids have been mainly performed by titration with perchloric acid [2] and sodium thiosulfate as titrants [3], spectrophotometry [4], polarography [5], and high-performance liquid chromatography (HPLC) [6–10]. Recently, Dmitrienko et al. [11] preconcentrated methylxanthines with solid phase extraction based on hyper-cross-linked polystyrene, followed HPLC characterization. The limit of detection (LOD) for DPP is 2 ng mL 1. Although HPLC is sensitive and accurate, it is also time-consuming, rather complicated, and expensive. Thus, a rapid and sensitive method for DPP detection must be developed. The fluorimetric probe method has been increasingly employed for the determination of pharmaceutical drugs because of its simplicity, sensitivity, and selectivity [12,13]. Quantum dots (QDs) or semiconductor nanoparticles have attracted much attention in
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Corresponding author. Tel.: þ 86 579 82287721. E-mail address: [email protected] (W. Wang).
0022-2313/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jlumin.2013.08.023
both fundamental and technical applications because of their unique size-tunable optical properties [14]. Compared with traditional organic dyes, QDs have many attractive features, such as broad excitation spectrum, narrow emission spectrum, high photobleaching threshold, and excellent photostability [15,16].Over the past years, considerable efforts have been made to develop a general means for chemical sensing with QDs as fluorescence probes for the determination of organic compounds and organic elementals, such as paeonol [17], gemifloxacin [18], roxithromycin [19], josamycin [20], dopamine, epinephrine [21], and vanadium [22]. Glutathione (GSH) is a very good ligand candidate for QD synthesis [23]. GSH-coated QDs are very stable in water solution and have high quantum yield and biocompatibility. GSH-capped QDs have been applied for the ultrasensitive detection of Pb2 þ [24], arsenic(III) [25], chromium(VI) [26], and cytochrome C [27]. In the present study, we successfully synthesized GSH–CdTe QDs and investigated their interaction with DPP. The fluorescence intensity of GSH–CdTe QDs was quenched in the presence of DPP in KH2PO4– Na2HPO4. The quenched intensity of fluorescence was proportional to the concentration of DPP. Based on this phenomenon, a new fluorescence-based method for the determination of DPP was developed. The proposed method was applied for the detection of DPP in human serum with satisfactory results. 2. Experimental 2.1. Apparatus All fluorescence measurements were performed with a LS-45 spectrophotofluorometer (Perkin-Elmer, USA) equipped with a
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1 cm quartz cell under ambient conditions, the slit-width for excitation and emission was both set at 10 nm. The absorption spectrum was acquired on a Lamda 950 UV–vis spectrophotometer (Perkin-Elmer, USA). All pH measurements were conducted with a model pHS-3C (Leici, Shanghai, China). Magnetic stirring was carried out using a HJ-3 magnetic stirrer (Zhengzhou, China).
diprophylline (1.0 10–3 mol L 1) were prepared with deionized water and stored at 4 1C. Deionized water was purified by employing a Milli-Q equipment from Millipore (Bedford, MA, USA) and used throughout this work.
2.2. Materials
NaHTe was used as Te precursor for CdTe QDs synthesis. It was prepared as described in previous report [28] with slight modifications. Briefly, 0.040 g Te and 0.0875 g NaBH4 were transferred into two neck flask under N2-flow. Then 0.4 mL deoxygenized water was injected into the reaction flask. The mixture was stirred for 2 hours under 35 1C, then the color of the mixture turned to pink then to white. At last the solution became a clear transparent solution and injected 11.6 mL deoxygenized water stalled for 1.5 h. The final solution was used for further experimentation.
Standard of diprophylline (DPP) was obtained from Shanghai Boyun Biotech. Co. Ltd, China. Sodium borohydride (96%), tellurium powder (99%), CdCl2 2.5H2O (96%), glutathione (GSH) were purchased from Aladdin Chemistry Co. Ltd (Shanghai China). Absolute ethanol was purchased from Jin hua Pharmaceutical Company (Jinhua China). All the other chemicals and materials used in the experiments were of analytical grade. The stock solution of
2.3. Preparation of NaHTe
Fig. 1. (A) UV–vis absorption and (B) FL spectea (excited at 350 nm) of GSH–CdTe QDs with different refluxing time. From a to i:1 h, 2 h, 3 h, 4 h, 5 h, 6 h.
S. Ying et al. / Journal of Luminescence 145 (2014) 575–581
2.4. Preparation of CdTe QDs CdTe QDs capped with GSH were prepared similar to the QDs capped with the other stabilizers [23]. 0.1790 g CdCl2 2.5H2O was dissolved in 50 mL of water and 0.2890 g GSH stabilizer was added under stirring. Then 1.0 mol L 1 NaOH was added dropwise to adjust the pH to 11.4. The solution was placed in a three-necked flask and deaerated by N2 for a while. Then 6.00 mL NaHTe was injected to the reaction solution. After the injection, the temperature was adjusted to 100 1C and kept for 5 hours under N2-flow with stiriing. The molar ratio of Cd2 þ :GSH:Te2 was settled as 5:6:1 [29]. After the nanocrystals reached desired size, the as-synthesized CdTe were precipitated
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by absolute ethanol followed by centrifugation at 16000 rpm for 10 min at room temperature, then the precipitate was purified by absolute ethanol three times in order to remove the excess GSH molecules. Finally, the precipitate was dissolved in ultrapure water homogeneously. 2.5. Procedures A quantity of 6 μL of GSH–CdTe QDs, an appropriate volume of DPP were added into a 6.0 mL colorimetric cylinder, then diluted with KH2PO4–Na2HPO4 medium and mixed thoroughly. The fluorescence spectra was obtained by scanning the emission from 450 nm to
Fig. 2. Effect of buffer solutions on the fluorescence intensity of GSH–CdTe C QDs ¼ 2.8 10–6 mol L 1, from a to c: KH2PO4–Na2HPO4, Tris–HCl, KH2PO4–Na2B4O7.
Fig. 3. Effect of buffer pH on the fluorescence intensity of GSH–CdTe- DPP system. C QDs ¼ 2.8 10–6 mol L 1, C DPP ¼ 1.67 10–6 mol L 1.
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650 nm on the spectrofluorimeter (excitation wavelength 350 nm, with 10 nm slit width both for excitation and emission), and the fluorescene intensity was recorded at the maximum emission wavelength. 2.6. Sample preparation Human serum samples were provided by the Hospital of Zhejiang Normal University (Jinhua, China). All samples were centrifuged for 10 min at 12,000 rpm to remove the plasma. The supernatant solution was collected and stored at 20 1C in the dark. A 1.00 mL aliquot of the sample underwent organic phase extraction by using 2.00 mL of trichloromethane–isopropanol (95:5 v/v) for 5 min. The sample was then subjected to centrifugation for 10 min at 4000 rpm. Finally, the organic/extract phase was evaporated to dryness by using a gentle stream of nitrogen at room temperature. The dried extract was dissolved in 100 μL of the buffer solution before analysis. For the recovery analysis, DPP at different levels were added before pre-purification.
3. Results and discussion 3.1. Characterization of GSH–CdTe QDs
(Fig. 3). F 0 and F are the fluorescence intensity of the aqueous GSH– CdTe QDs in the absence and presence of DPP, respectively. The reason that fluorescence quenching was critically dependent on the acidity of the solution might be the different surface conditions of GSH–CdTe QDs at different pH values. At lower pH level, the thiolate is protonated and detached from the QDs, which increases the defect-related surface state and decreases the fluorescence emission. Moreover, the surface of QDs is more sensitive to the quencher, and enhanced quenching effects are observed [34]. Fig. 3 also shows that the quenching percentage decreases when pH48.4. A possible explanation is that the surface of CdTe QDs was coated by excessive Cd(OH)2 and CdTe/Cd (OH)2, and the core/shell QDs appeared [23]. However, the quenching percentage decreases because of the dominating electrostatic repulsion between DPP and QDs at a higher pH. Therefore, the phosphate buffer solution at pH 8.4 was chosen in this study to provide an optimal condition for maximal quenching percentage. 3.2.2. Effect of temperature The solution temperature is one of the key factors that affect the fluorescence intensity of CdTe QDs. The fluorescence intensity of CdTe QDs was recorded with varying temperatures from 25 1C to 65 1C, and the results are shown in Fig. 4. The relationship between temperature and fluorescence intensity of CdTe QDs (Fig. 4B) is nearly linear (negative). Similar results were also observed by Yu et al. [35] and
Fig. 1 presents the typical absorption and fluorescence spectra of the prepared GSH–CdTe QDs with different refluxing times. Fig. 1A shows that the absorption spectrum of CdTe QDs was not a singlepeak absorption but a broad absorption; thus, different sizes of QDs can be stimulated with the same monochromatic light source. The absorption peak shifts toward longer wavelengths with prolonged refluxing times, which clearly indicates a gradual increase in particle size [30]. Based on the UV–vis absorption spectra, the particle sizes of the QDs were around 2.04 nm, 2.60 nm, 2.85 nm, 3.06 nm, 3.21 nm, and 3.30 nm, respectively, by using the following empirical equation [31]: D ¼ ð9:8127 107 Þλ ð1:7147 103 Þλ þ 1:0064λ194:84 3
2
where D (nm) is the diameter of a given QD, and λ (nm) is the wavelength of the first excitonic absorption peak of the UV–vis absorption spectrum. Fig. 1B shows that the line width of the fluorescence spectrum is narrow, which shows that the as-prepared CdTe QDs were nearly monodisperse and homogenous [32]. The fluorescence intensity shows a rising tendency with refluxing time from 1 h to 3 h, followed by a significant decrease when the refluxing time was further increased to 6 h. The maximum fluorescence intensity was observed at a reflux time of 3 h. This result indicates that the QDs gradually grew at a controlled speed. The results are attributed to the changes in size and constitution of QDs during growth. In addition, the peak wavelength gradually moves to the red region of the spectrum, which indicates that the size of the QDs gradually increases with increased refluxing time.
Fig. 4. Effect of temperature on the fluorescence intensity of CdTe QDs, from a to e: 25 1C, 35 1C, 45 1C, 55 1C, 65 1C.
3.2. Optimization of reaction conditions 3.2.1. Effect of buffer solution The effect of different buffer solutions, such as KH2PO4–Na2HPO4, Tris–HCl, and KH2PO4–Na2B4O7, on the fluorescence intensity of GSH– CdTe QDs were tested (Fig. 2), and the maximum fluorescence intensity was obtained when KH2PO4–Na2HPO4 was used as the buffer. Previous reports have suggested that the pH of the buffer solution has an important function in the interaction between QDs and other molecules [33]. The effect of pH on the fluorescence intensity of the interaction between CdTe QDs and DPP was also investigated. The maximum value of ðF 0 FÞ is obtained at pH 8.4
Fig. 5. Effect of incubation time on the absorption of GSH–CdTe–DPP system. C QDs ¼11.2 10–6 mol L 1, C DPP ¼6.67 10–6 mol L 1.
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Peng et al. [19]. The calculated value of the temperature coefficient Tcoef confirmed that the non-radiant deactivation process involves internal conversion when jTcoef jr1% [36]. The value obtained for CdTe QDs is 0.86%. Thus, the non-radiant deactivation process occurred for CdTe QDs. Therefore, the decreased quantum yield of CdTe QDs with increasing temperature is attributed to the decreasing fluorescence intensity when they are excited with the same energy. The maximal fluorescence intensity is reached when the solution temperature is 25 1C. Therefore, an incubation temperature of 25 1C was adopted in this work. 3.2.3. Effect of incubation time and adding sequence The kinetic characteristics of the reaction system were investigated at 25 1C (Fig. 5). Upon the addition of DPP (6.67 10–6 mol L 1) to QDs (11.2 10–6 mol L 1), a stable absorption signal at 270 nm can be obtained within a reaction time of about 8 min and remained constant for at least 8 min. Hence, the reaction time of 8 min was adopted, and UV measurements were completed within 16 min in the following experiments. The mixing sequences of aqueous GSH–CdTe QDs, DPP, and the buffer solution were investigated by recording the fluorescence intensity of the aqueous GSH–CdTe QDs–DPP solution system with GSH–CdTe QDs and DPP concentrations of 11.2 10–6 and 6.67 10– 6 mol L 1, respectively. The results show that the different adding sequences had little influence on the fluorescence intensity of aqueous GSH–CdTe QDs–DPP solution system. Similar results were also observed by Dong et al. [17]. The repeatability of the assay result was also influenced by the adding sequence of the reagents. Credible and stable results can be obtained according to the following sequence: the DPP solution was added to GSH–CdTe QDs, the buffer solution was then added, and the solution was mixed thoroughly. 3.3. Calibration curve and detection limit In optimized experimental conditions, the fluorescence spectrum of aqueous GSH–CdTe QDs with different concentrations of DPP was
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obtained, and the results are shown in Fig. 6. The results indicate that the fluorescence intensity of the GSH–CdTe QDs significantly decreased with increasing DPP concentration (C DPP ). The relationship between F 0 =F and C DPP from 1.67 10–6 mol L 1 to 1.33 10– 5 mol L 1 can be described by a linear equation (correlation coefficient: 0.9970) as follows: F 0 =F ¼ 1:0662 þ 3:8238 104 C DPP The LOD for DPP was 2.24 10–7 mol L 1 (S/N ¼ 3). The relative standard deviation for six determinations of 1.67 10–6 mol L 1 DPP was 3.05%. This result shows that the method has good accuracy and precision for the detection of DPP.
Table 1 Interference of coexistence substance on the FL intensity of GSH–CdTe QDs with DPP. Foreign substance
Times of tolerance
Change in ΔF(%)
Na þ Kþ Cl NO3 SO42 Br Glucose
50 100 100 100 60 100 50
4.22 1.56 1.56 1 0.92 1.56 1.36
Table 2 Analytical results for detection of DPP in samples (n¼ 3). Samples DPP in samples (10–6 mol L 1)
NFa NF NF
1 2 3 a
DPP added (10– 6 mol L 1) 1.67 3.33 5.00
DPP found (10– mol L 1)
Average recovery (%)
RSDs (%)
1.46 3.30 5.90
87.41 99.04 117.94
2.89 2.36 1.51
6
Not found.
Fig. 6. Fluorescence quenching of aqueous GSH–CdTe QDs by different amounts of DPP. C QDs ¼ 2.8 10–6 mol L 1, C DPP (from a to i): 0, 1.67, 3.33, 5, 6.67, 8.33, 10, 11.67, 13.33 10–6 mol L 1.
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3.4. Interference of co-existing foreign substances
3.6. Possible quenching mechanism
Many compounds have been reported to have potential to quench the fluorescence intensity of QDs [37]. To investigate the possibility of a practical application of the proposed method, the effect of coexisting foreign substances on the determination of DPP was investigated in optimum conditions. Table 1 describes the influence of coexisting substances on the fluorescence intensity of GSH–CdTe QDs. A substance that gave a relative change in fluorescence intensity less than 5% was considered as non-interference in the assay. Most of the common ions did not produce noticeable effects on the fluorescence intensity of GSH–CdTe QDs (Table 1). The tolerance limits of these substances were at least 50 times than the DPP amount. The interactions between QDs and other biological substances were not tested in this work based on the results of previous works [19,27,38]. Cao et al. [27] further investigated the fluorescence responses of GSH– CdTe QDs to common biological substances, such as several proteins and amino acids. Their results showed that most of the tested substances did not exhibit any significant effect on the photoluminescence emission of the QDs even at relatively high concentrations. Chen et al. [38] also reported that the interferences from the components of human serum, such as glycine, isoleucine, aspartic acid, and stearic acid, could be neglected because of their low concentration in the samples and in subsequent dilutions.
The quenching mechanism of QDs may be due to the inner filter effect, Förster resonance energy transfer, surface molecular charge in QDs, and charge diverting [39]. To explore the mechanisms of the reaction, the UV–vis absorption spectra of GSH–CdTe QDs were investigated in the absence and presence of DPP. As shown in Fig. 7, DPP has one absorption peak at 270 nm, whereas GSH–CdTe QDs has a weak absorption in the wavelength range of 450 nm to 600 nm. Thus, the quenching effect was not due to an inner filter that is caused by the absorption of the emission wavelength by DPP. The Förster resonance energy transfer is a process that involves nonradiative energy transfer from a photon-excited donor molecule, after capturing a higher energy photon, to an acceptor molecule of a different species, which may relax to its ground state by emitting a lower energy photon. No overlaps were observed between the fluorescence spectra
3.5. Analytical application The proposed method was applied for the detection of DPP in human serum in optimized conditions. Various amounts of freshly prepared analytes of the sample solution and GSH–CdTe QDs (2.8 10–6 mol L 1) were diluted with 3.00 mL of the buffer solution, and the fluorescent spectra were obtained after 8 min. The result shows that no DPP was detected in the human serum samples. To calculate for the recovery efficiency of this method, DPP at different spiking levels (1.67, 3.33, and 5.00 10–6 mol L 1) were added to the sample solutions before pre-purification. Table 2 shows that the recoveries of the method were in the range of 87.41% to 117.94% with a relative standard deviation less than 2.89%.
Fig. 8. Fluorescence decay as a function of time (ns) for GSH–CdTe QDs before and after adding DPP at pH 8.4 KH2PO4–Na2HPO4 buffer solution. (excitation wavelength was 350 nm). (CQDs¼ 2.8 10–6 mol L 1, C DPP ¼3.33, 6.67 10–6 mol L 1 respectively).
Fig. 7. The absorption spectra of (a) GSH–CdTe QDs–DPP system(a); (b) GSH–CdTe QDs; (c) DPP. C DPP :6.67 10–6 mol L 1, C QDs :18.7 10–6 mol L 1.
S. Ying et al. / Journal of Luminescence 145 (2014) 575–581
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Scheme 1. Mechanism of fluorescence quenching process.
band of GSH–CdTe QDs and the absorption spectra band of DPP that could lead to the Förster resonance energy transfer. In addition, no obvious change was observed for the QDs absorption spectra before and after adding DPP (Fig. 7A and B). A blue- or red-shift of the fluorescence emission spectra (Fig. 6) was also not observed when the concentrations of DPP were changed, which also means that GSH– CdTe QDs do not aggregate or become smaller after adding DPP [40]. For further investigation, the fluorescence lifetimes of QDs in the absence and presence of DPP were measured. As seen in Fig. 8, the fluorescence decay profile for GSH–CdTe QDs remains unchanged with addition of DPP. The measured fluorescence decay time for the GSH–CdTe QDs and GSH–CdTe QDs–DPP system were 38.80 and 38.03 ns, respectively. The results imply that the observed quenching process occurs by a static mechanism [41]. The fluorescence of GSH– CdTe QDs originates from the recombination of an electron and a hole [39]. As an effective quencher for the GSH–CdTe QDs, DPP may function as an electron acceptor for the conduction band electron from the QDs and disrupts the radioactive recombination process and quenches the fluorescence [42]. The possible quenching process is shown in Scheme 1. Based on the above analysis, the fast electron transfer from QDs to DPP is considered to be the potential cause of the fluorescence quenching for the systems, whereas the exact quenching mechanism awaits further investigations. 4. Conclusions A novel and convenient method for DPP analysis was developed based on the fluorescence quenching of GSH–CdTe QDs by using DPP. In optimum conditions, a linear relationship between the fluorescence intensity ratio of the system and the concentration of DPP ranging from 1.67 10–6 mol L 1 to 1.33 10–5 mol L 1 can be achieved. DPP can be detected with this simple strategy with a high sensitivity. The possible quenching mechanism is due to the fast electron transfer from QDs to DPP. The method was applied to detect DPP in human serum samples with satisfactory results. The assay is characterized by its simplicity, short analysis time, less sample consumption, and fewer reagent requirements. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21175118) and Zhejiang Normal University (No. ZC304007104 ).
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Simple and sensitive detection method for diprophylline using glutathione-capped CdTe quantum dots as fluorescence probes Suyan Ying a, Shumin Cui a, Weiping Wang a,n, Jiuju Feng a, Jianrong Chen b a b
College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, China College of Geography and Environmental Science, Zhejiang Normal University, Jinhua 321004, China
art ic l e i nf o
a b s t r a c t
Article history: Received 18 October 2012 Received in revised form 4 August 2013 Accepted 14 August 2013 Available online 24 August 2013
A simple and sensitive method for detecting diprophylline (DPP) was developed based on the fluorescence quenching of glutathione-capped CdTe quantum dots (GSH–CdTe QDs) by using diprophylline in a KH2PO4–Na2HPO4 medium. Parameters affecting the quenching efficiency, including types and pH of buffer solutions as well as temperature, reaction time, adding sequence, and interfering substances, were investigated and optimized. In optimum conditions, the calibration plot of the quenched fluorescence intensity F 0 =F with a DPP concentration range of 1.67 10–6 mol L 1 to 1.33 10–5 mol L 1 –7 was linear. The detection limit (with signal to noise ratio of 3) for DPP was 2.24 10 mol L 1. The proposed method was successfully applied for detecting DPP in human serum. The recovery of the method was in the range of 87.41% to 117.94%. Finally, the possible quenching mechanism of GSH–CdTe QDs and DPP was also discussed. & 2013 Elsevier B.V. All rights reserved.
Keywords: Glutathione-capped CdTe quantum dots Diprophylline Fluorescence quenching
1. Introduction Diprophylline (DPP) is a methylxanthine drug that can directly relax the smooth bronchi muscles and pulmonary blood vessels. It is usually employed for the symptomatic relief or clinical prevention of bronchial asthma and chronic obstructive pulmonary disease [1]. Compared with theophylline, DPP has an enhanced water solubility and fewer adverse reactions. The main metabolites of DPP in blood are its prototype and theophylline derivatives. After 24 h of medication, 83% of DPP is excreted through urine. However, the transport and metabolic process of DPP in vivo remain unclear. Wang et al. [1] investigated the binding characteristic of DPP with lysozyme via spectroscopic methods. Until now, the detection and quantification of DPP in biological fluids have been mainly performed by titration with perchloric acid [2] and sodium thiosulfate as titrants [3], spectrophotometry [4], polarography [5], and high-performance liquid chromatography (HPLC) [6–10]. Recently, Dmitrienko et al. [11] preconcentrated methylxanthines with solid phase extraction based on hyper-cross-linked polystyrene, followed HPLC characterization. The limit of detection (LOD) for DPP is 2 ng mL 1. Although HPLC is sensitive and accurate, it is also time-consuming, rather complicated, and expensive. Thus, a rapid and sensitive method for DPP detection must be developed. The fluorimetric probe method has been increasingly employed for the determination of pharmaceutical drugs because of its simplicity, sensitivity, and selectivity [12,13]. Quantum dots (QDs) or semiconductor nanoparticles have attracted much attention in
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Corresponding author. Tel.: þ 86 579 82287721. E-mail address: [email protected] (W. Wang).
0022-2313/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jlumin.2013.08.023
both fundamental and technical applications because of their unique size-tunable optical properties [14]. Compared with traditional organic dyes, QDs have many attractive features, such as broad excitation spectrum, narrow emission spectrum, high photobleaching threshold, and excellent photostability [15,16].Over the past years, considerable efforts have been made to develop a general means for chemical sensing with QDs as fluorescence probes for the determination of organic compounds and organic elementals, such as paeonol [17], gemifloxacin [18], roxithromycin [19], josamycin [20], dopamine, epinephrine [21], and vanadium [22]. Glutathione (GSH) is a very good ligand candidate for QD synthesis [23]. GSH-coated QDs are very stable in water solution and have high quantum yield and biocompatibility. GSH-capped QDs have been applied for the ultrasensitive detection of Pb2 þ [24], arsenic(III) [25], chromium(VI) [26], and cytochrome C [27]. In the present study, we successfully synthesized GSH–CdTe QDs and investigated their interaction with DPP. The fluorescence intensity of GSH–CdTe QDs was quenched in the presence of DPP in KH2PO4– Na2HPO4. The quenched intensity of fluorescence was proportional to the concentration of DPP. Based on this phenomenon, a new fluorescence-based method for the determination of DPP was developed. The proposed method was applied for the detection of DPP in human serum with satisfactory results. 2. Experimental 2.1. Apparatus All fluorescence measurements were performed with a LS-45 spectrophotofluorometer (Perkin-Elmer, USA) equipped with a
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1 cm quartz cell under ambient conditions, the slit-width for excitation and emission was both set at 10 nm. The absorption spectrum was acquired on a Lamda 950 UV–vis spectrophotometer (Perkin-Elmer, USA). All pH measurements were conducted with a model pHS-3C (Leici, Shanghai, China). Magnetic stirring was carried out using a HJ-3 magnetic stirrer (Zhengzhou, China).
diprophylline (1.0 10–3 mol L 1) were prepared with deionized water and stored at 4 1C. Deionized water was purified by employing a Milli-Q equipment from Millipore (Bedford, MA, USA) and used throughout this work.
2.2. Materials
NaHTe was used as Te precursor for CdTe QDs synthesis. It was prepared as described in previous report [28] with slight modifications. Briefly, 0.040 g Te and 0.0875 g NaBH4 were transferred into two neck flask under N2-flow. Then 0.4 mL deoxygenized water was injected into the reaction flask. The mixture was stirred for 2 hours under 35 1C, then the color of the mixture turned to pink then to white. At last the solution became a clear transparent solution and injected 11.6 mL deoxygenized water stalled for 1.5 h. The final solution was used for further experimentation.
Standard of diprophylline (DPP) was obtained from Shanghai Boyun Biotech. Co. Ltd, China. Sodium borohydride (96%), tellurium powder (99%), CdCl2 2.5H2O (96%), glutathione (GSH) were purchased from Aladdin Chemistry Co. Ltd (Shanghai China). Absolute ethanol was purchased from Jin hua Pharmaceutical Company (Jinhua China). All the other chemicals and materials used in the experiments were of analytical grade. The stock solution of
2.3. Preparation of NaHTe
Fig. 1. (A) UV–vis absorption and (B) FL spectea (excited at 350 nm) of GSH–CdTe QDs with different refluxing time. From a to i:1 h, 2 h, 3 h, 4 h, 5 h, 6 h.
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2.4. Preparation of CdTe QDs CdTe QDs capped with GSH were prepared similar to the QDs capped with the other stabilizers [23]. 0.1790 g CdCl2 2.5H2O was dissolved in 50 mL of water and 0.2890 g GSH stabilizer was added under stirring. Then 1.0 mol L 1 NaOH was added dropwise to adjust the pH to 11.4. The solution was placed in a three-necked flask and deaerated by N2 for a while. Then 6.00 mL NaHTe was injected to the reaction solution. After the injection, the temperature was adjusted to 100 1C and kept for 5 hours under N2-flow with stiriing. The molar ratio of Cd2 þ :GSH:Te2 was settled as 5:6:1 [29]. After the nanocrystals reached desired size, the as-synthesized CdTe were precipitated
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by absolute ethanol followed by centrifugation at 16000 rpm for 10 min at room temperature, then the precipitate was purified by absolute ethanol three times in order to remove the excess GSH molecules. Finally, the precipitate was dissolved in ultrapure water homogeneously. 2.5. Procedures A quantity of 6 μL of GSH–CdTe QDs, an appropriate volume of DPP were added into a 6.0 mL colorimetric cylinder, then diluted with KH2PO4–Na2HPO4 medium and mixed thoroughly. The fluorescence spectra was obtained by scanning the emission from 450 nm to
Fig. 2. Effect of buffer solutions on the fluorescence intensity of GSH–CdTe C QDs ¼ 2.8 10–6 mol L 1, from a to c: KH2PO4–Na2HPO4, Tris–HCl, KH2PO4–Na2B4O7.
Fig. 3. Effect of buffer pH on the fluorescence intensity of GSH–CdTe- DPP system. C QDs ¼ 2.8 10–6 mol L 1, C DPP ¼ 1.67 10–6 mol L 1.
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650 nm on the spectrofluorimeter (excitation wavelength 350 nm, with 10 nm slit width both for excitation and emission), and the fluorescene intensity was recorded at the maximum emission wavelength. 2.6. Sample preparation Human serum samples were provided by the Hospital of Zhejiang Normal University (Jinhua, China). All samples were centrifuged for 10 min at 12,000 rpm to remove the plasma. The supernatant solution was collected and stored at 20 1C in the dark. A 1.00 mL aliquot of the sample underwent organic phase extraction by using 2.00 mL of trichloromethane–isopropanol (95:5 v/v) for 5 min. The sample was then subjected to centrifugation for 10 min at 4000 rpm. Finally, the organic/extract phase was evaporated to dryness by using a gentle stream of nitrogen at room temperature. The dried extract was dissolved in 100 μL of the buffer solution before analysis. For the recovery analysis, DPP at different levels were added before pre-purification.
3. Results and discussion 3.1. Characterization of GSH–CdTe QDs
(Fig. 3). F 0 and F are the fluorescence intensity of the aqueous GSH– CdTe QDs in the absence and presence of DPP, respectively. The reason that fluorescence quenching was critically dependent on the acidity of the solution might be the different surface conditions of GSH–CdTe QDs at different pH values. At lower pH level, the thiolate is protonated and detached from the QDs, which increases the defect-related surface state and decreases the fluorescence emission. Moreover, the surface of QDs is more sensitive to the quencher, and enhanced quenching effects are observed [34]. Fig. 3 also shows that the quenching percentage decreases when pH48.4. A possible explanation is that the surface of CdTe QDs was coated by excessive Cd(OH)2 and CdTe/Cd (OH)2, and the core/shell QDs appeared [23]. However, the quenching percentage decreases because of the dominating electrostatic repulsion between DPP and QDs at a higher pH. Therefore, the phosphate buffer solution at pH 8.4 was chosen in this study to provide an optimal condition for maximal quenching percentage. 3.2.2. Effect of temperature The solution temperature is one of the key factors that affect the fluorescence intensity of CdTe QDs. The fluorescence intensity of CdTe QDs was recorded with varying temperatures from 25 1C to 65 1C, and the results are shown in Fig. 4. The relationship between temperature and fluorescence intensity of CdTe QDs (Fig. 4B) is nearly linear (negative). Similar results were also observed by Yu et al. [35] and
Fig. 1 presents the typical absorption and fluorescence spectra of the prepared GSH–CdTe QDs with different refluxing times. Fig. 1A shows that the absorption spectrum of CdTe QDs was not a singlepeak absorption but a broad absorption; thus, different sizes of QDs can be stimulated with the same monochromatic light source. The absorption peak shifts toward longer wavelengths with prolonged refluxing times, which clearly indicates a gradual increase in particle size [30]. Based on the UV–vis absorption spectra, the particle sizes of the QDs were around 2.04 nm, 2.60 nm, 2.85 nm, 3.06 nm, 3.21 nm, and 3.30 nm, respectively, by using the following empirical equation [31]: D ¼ ð9:8127 107 Þλ ð1:7147 103 Þλ þ 1:0064λ194:84 3
2
where D (nm) is the diameter of a given QD, and λ (nm) is the wavelength of the first excitonic absorption peak of the UV–vis absorption spectrum. Fig. 1B shows that the line width of the fluorescence spectrum is narrow, which shows that the as-prepared CdTe QDs were nearly monodisperse and homogenous [32]. The fluorescence intensity shows a rising tendency with refluxing time from 1 h to 3 h, followed by a significant decrease when the refluxing time was further increased to 6 h. The maximum fluorescence intensity was observed at a reflux time of 3 h. This result indicates that the QDs gradually grew at a controlled speed. The results are attributed to the changes in size and constitution of QDs during growth. In addition, the peak wavelength gradually moves to the red region of the spectrum, which indicates that the size of the QDs gradually increases with increased refluxing time.
Fig. 4. Effect of temperature on the fluorescence intensity of CdTe QDs, from a to e: 25 1C, 35 1C, 45 1C, 55 1C, 65 1C.
3.2. Optimization of reaction conditions 3.2.1. Effect of buffer solution The effect of different buffer solutions, such as KH2PO4–Na2HPO4, Tris–HCl, and KH2PO4–Na2B4O7, on the fluorescence intensity of GSH– CdTe QDs were tested (Fig. 2), and the maximum fluorescence intensity was obtained when KH2PO4–Na2HPO4 was used as the buffer. Previous reports have suggested that the pH of the buffer solution has an important function in the interaction between QDs and other molecules [33]. The effect of pH on the fluorescence intensity of the interaction between CdTe QDs and DPP was also investigated. The maximum value of ðF 0 FÞ is obtained at pH 8.4
Fig. 5. Effect of incubation time on the absorption of GSH–CdTe–DPP system. C QDs ¼11.2 10–6 mol L 1, C DPP ¼6.67 10–6 mol L 1.
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Peng et al. [19]. The calculated value of the temperature coefficient Tcoef confirmed that the non-radiant deactivation process involves internal conversion when jTcoef jr1% [36]. The value obtained for CdTe QDs is 0.86%. Thus, the non-radiant deactivation process occurred for CdTe QDs. Therefore, the decreased quantum yield of CdTe QDs with increasing temperature is attributed to the decreasing fluorescence intensity when they are excited with the same energy. The maximal fluorescence intensity is reached when the solution temperature is 25 1C. Therefore, an incubation temperature of 25 1C was adopted in this work. 3.2.3. Effect of incubation time and adding sequence The kinetic characteristics of the reaction system were investigated at 25 1C (Fig. 5). Upon the addition of DPP (6.67 10–6 mol L 1) to QDs (11.2 10–6 mol L 1), a stable absorption signal at 270 nm can be obtained within a reaction time of about 8 min and remained constant for at least 8 min. Hence, the reaction time of 8 min was adopted, and UV measurements were completed within 16 min in the following experiments. The mixing sequences of aqueous GSH–CdTe QDs, DPP, and the buffer solution were investigated by recording the fluorescence intensity of the aqueous GSH–CdTe QDs–DPP solution system with GSH–CdTe QDs and DPP concentrations of 11.2 10–6 and 6.67 10– 6 mol L 1, respectively. The results show that the different adding sequences had little influence on the fluorescence intensity of aqueous GSH–CdTe QDs–DPP solution system. Similar results were also observed by Dong et al. [17]. The repeatability of the assay result was also influenced by the adding sequence of the reagents. Credible and stable results can be obtained according to the following sequence: the DPP solution was added to GSH–CdTe QDs, the buffer solution was then added, and the solution was mixed thoroughly. 3.3. Calibration curve and detection limit In optimized experimental conditions, the fluorescence spectrum of aqueous GSH–CdTe QDs with different concentrations of DPP was
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obtained, and the results are shown in Fig. 6. The results indicate that the fluorescence intensity of the GSH–CdTe QDs significantly decreased with increasing DPP concentration (C DPP ). The relationship between F 0 =F and C DPP from 1.67 10–6 mol L 1 to 1.33 10– 5 mol L 1 can be described by a linear equation (correlation coefficient: 0.9970) as follows: F 0 =F ¼ 1:0662 þ 3:8238 104 C DPP The LOD for DPP was 2.24 10–7 mol L 1 (S/N ¼ 3). The relative standard deviation for six determinations of 1.67 10–6 mol L 1 DPP was 3.05%. This result shows that the method has good accuracy and precision for the detection of DPP.
Table 1 Interference of coexistence substance on the FL intensity of GSH–CdTe QDs with DPP. Foreign substance
Times of tolerance
Change in ΔF(%)
Na þ Kþ Cl NO3 SO42 Br Glucose
50 100 100 100 60 100 50
4.22 1.56 1.56 1 0.92 1.56 1.36
Table 2 Analytical results for detection of DPP in samples (n¼ 3). Samples DPP in samples (10–6 mol L 1)
NFa NF NF
1 2 3 a
DPP added (10– 6 mol L 1) 1.67 3.33 5.00
DPP found (10– mol L 1)
Average recovery (%)
RSDs (%)
1.46 3.30 5.90
87.41 99.04 117.94
2.89 2.36 1.51
6
Not found.
Fig. 6. Fluorescence quenching of aqueous GSH–CdTe QDs by different amounts of DPP. C QDs ¼ 2.8 10–6 mol L 1, C DPP (from a to i): 0, 1.67, 3.33, 5, 6.67, 8.33, 10, 11.67, 13.33 10–6 mol L 1.
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3.4. Interference of co-existing foreign substances
3.6. Possible quenching mechanism
Many compounds have been reported to have potential to quench the fluorescence intensity of QDs [37]. To investigate the possibility of a practical application of the proposed method, the effect of coexisting foreign substances on the determination of DPP was investigated in optimum conditions. Table 1 describes the influence of coexisting substances on the fluorescence intensity of GSH–CdTe QDs. A substance that gave a relative change in fluorescence intensity less than 5% was considered as non-interference in the assay. Most of the common ions did not produce noticeable effects on the fluorescence intensity of GSH–CdTe QDs (Table 1). The tolerance limits of these substances were at least 50 times than the DPP amount. The interactions between QDs and other biological substances were not tested in this work based on the results of previous works [19,27,38]. Cao et al. [27] further investigated the fluorescence responses of GSH– CdTe QDs to common biological substances, such as several proteins and amino acids. Their results showed that most of the tested substances did not exhibit any significant effect on the photoluminescence emission of the QDs even at relatively high concentrations. Chen et al. [38] also reported that the interferences from the components of human serum, such as glycine, isoleucine, aspartic acid, and stearic acid, could be neglected because of their low concentration in the samples and in subsequent dilutions.
The quenching mechanism of QDs may be due to the inner filter effect, Förster resonance energy transfer, surface molecular charge in QDs, and charge diverting [39]. To explore the mechanisms of the reaction, the UV–vis absorption spectra of GSH–CdTe QDs were investigated in the absence and presence of DPP. As shown in Fig. 7, DPP has one absorption peak at 270 nm, whereas GSH–CdTe QDs has a weak absorption in the wavelength range of 450 nm to 600 nm. Thus, the quenching effect was not due to an inner filter that is caused by the absorption of the emission wavelength by DPP. The Förster resonance energy transfer is a process that involves nonradiative energy transfer from a photon-excited donor molecule, after capturing a higher energy photon, to an acceptor molecule of a different species, which may relax to its ground state by emitting a lower energy photon. No overlaps were observed between the fluorescence spectra
3.5. Analytical application The proposed method was applied for the detection of DPP in human serum in optimized conditions. Various amounts of freshly prepared analytes of the sample solution and GSH–CdTe QDs (2.8 10–6 mol L 1) were diluted with 3.00 mL of the buffer solution, and the fluorescent spectra were obtained after 8 min. The result shows that no DPP was detected in the human serum samples. To calculate for the recovery efficiency of this method, DPP at different spiking levels (1.67, 3.33, and 5.00 10–6 mol L 1) were added to the sample solutions before pre-purification. Table 2 shows that the recoveries of the method were in the range of 87.41% to 117.94% with a relative standard deviation less than 2.89%.
Fig. 8. Fluorescence decay as a function of time (ns) for GSH–CdTe QDs before and after adding DPP at pH 8.4 KH2PO4–Na2HPO4 buffer solution. (excitation wavelength was 350 nm). (CQDs¼ 2.8 10–6 mol L 1, C DPP ¼3.33, 6.67 10–6 mol L 1 respectively).
Fig. 7. The absorption spectra of (a) GSH–CdTe QDs–DPP system(a); (b) GSH–CdTe QDs; (c) DPP. C DPP :6.67 10–6 mol L 1, C QDs :18.7 10–6 mol L 1.
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Scheme 1. Mechanism of fluorescence quenching process.
band of GSH–CdTe QDs and the absorption spectra band of DPP that could lead to the Förster resonance energy transfer. In addition, no obvious change was observed for the QDs absorption spectra before and after adding DPP (Fig. 7A and B). A blue- or red-shift of the fluorescence emission spectra (Fig. 6) was also not observed when the concentrations of DPP were changed, which also means that GSH– CdTe QDs do not aggregate or become smaller after adding DPP [40]. For further investigation, the fluorescence lifetimes of QDs in the absence and presence of DPP were measured. As seen in Fig. 8, the fluorescence decay profile for GSH–CdTe QDs remains unchanged with addition of DPP. The measured fluorescence decay time for the GSH–CdTe QDs and GSH–CdTe QDs–DPP system were 38.80 and 38.03 ns, respectively. The results imply that the observed quenching process occurs by a static mechanism [41]. The fluorescence of GSH– CdTe QDs originates from the recombination of an electron and a hole [39]. As an effective quencher for the GSH–CdTe QDs, DPP may function as an electron acceptor for the conduction band electron from the QDs and disrupts the radioactive recombination process and quenches the fluorescence [42]. The possible quenching process is shown in Scheme 1. Based on the above analysis, the fast electron transfer from QDs to DPP is considered to be the potential cause of the fluorescence quenching for the systems, whereas the exact quenching mechanism awaits further investigations. 4. Conclusions A novel and convenient method for DPP analysis was developed based on the fluorescence quenching of GSH–CdTe QDs by using DPP. In optimum conditions, a linear relationship between the fluorescence intensity ratio of the system and the concentration of DPP ranging from 1.67 10–6 mol L 1 to 1.33 10–5 mol L 1 can be achieved. DPP can be detected with this simple strategy with a high sensitivity. The possible quenching mechanism is due to the fast electron transfer from QDs to DPP. The method was applied to detect DPP in human serum samples with satisfactory results. The assay is characterized by its simplicity, short analysis time, less sample consumption, and fewer reagent requirements. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21175118) and Zhejiang Normal University (No. ZC304007104 ).
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