Exploring Mn-Doped ZnS Quantum Dots for the Room-Temperature

Anal. Chem. 2008, 80, 3832–3837

Exploring Mn-Doped ZnS Quantum Dots for the Room-Temperature Phosphorescence Detection of Enoxacin in Biological Fluids Yu He, He-Fang Wang, and Xiu-Ping Yan* Research Center for Analytical Sciences, College of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, China While most research works focus on the development of quantum dots (QDs)-based fluorescence sensors, much less attention is paid to the phosphorescence properties of QDs and their potential for phosphorescence detection. In this work, the phosphorescence property of Mn-doped ZnS QDs is explored to develop a novel room-temperature phosphorescence (RTP) method for the facile, rapid, costeffective, sensitive, and selective detection of enoxacin in biological fluids. The Mn-doped ZnS QDs-based RTP method reported here does not need the use of deoxidants and other inducers and allows the detection of enoxacin in biological fluids without interference from autofluorescence and the scattering light of the matrix. The Mn-doped ZnS QDs offer excellent selectivity for detecting enoxacin in the presence of the main relevant metal ions in biological fluids, biomolecules, and other kinds of antibiotics. Quenching of the phosphorescence emission due to the addition of enoxacin at 1.0 µM is unaffected by 5000-fold excesses of Na+ and 10000-fold excesses of K+, Mg2+, and Ca2+. Amino acids such as tryptophan, histidine, and L-cysteine at 1000-fold concentration of enoxacin do not affect the detection of enoxacin. Glucose does not affect the detection at 10000-fold concentration of enoxacin. Typical coadministers (mainly other types of antibiotics) such as ceftezole, cefoperazone, oxacillin, and kalii dehydrographolidi succinas are permitted at 50-, 10-, 100-, and 50-fold excesses, respectively, without interference with the detection of enoxacin. The precision for 11 replicate detections of 0.4 µM enoxacin is 1.8% (RSD). The detection limit for enoxacin is 58.6 nM. The recovery of spiked enoxacin in human urine and serum samples ranges from 94 to 104%. The developed Mndoped ZnS QDs-based RTP method is employed to monitor the time-dependent concentration of enoxacin in urine from a healthy volunteer after the oral medication of enoxacin. The investigation provides evidence that doped QDs are promising for RTP detection in further applications. The exploration of systems capable of sensing and recognizing based on quantum dots (QDs) is a topic of considerable interest, since QDs offer advantages over conventional organic and inorganic fluorophores such as great photostability, high photo* Corresponding author. Fax: (86)22-23506075. E-mail: [email protected].

3832

Analytical Chemistry, Vol. 80, No. 10, May 15, 2008

luminescence efficiency, size-dependent emission wavelengths, and sharp emission profile.1 Subtle changes of the surface properties of QDs can result in dramatic changes in their optical properties. This feature of QDs offers many opportunities for detecting various specific analytes. So functionalized QDs have become one of the most important fluorescence probes for a great number of analytes including ions,2–4 biomacromolecules,5–8 and small molecules.9–11 While more and more research has been focusing on the exploration of QDs as fluorescence sensors, much less attention has been paid to the phosphorescence properties of QDs and their potential as phosphorescence sensors. Room-temperature phosphorescence (RTP) detection possesses many advantages over the fluorescence method. The long lifetime of phosphorescence allows an appropriate delay time, so that any fluorescence emission and scattering light can be easily avoided. The selectivity is enhanced because it is a less usual phenomenon than that of fluorescence.12 The delayed luminescence lifetimes of dopants and host semiconductors have been studied in a number of doped semiconductor systems.13–15 Doped semiconductors have also been employed as

(1) Lan, G. Y.; Lin, Y. W.; Huang, Y. F.; Chang, H. T. J. Mater. Chem. 2007, 17, 2661–2666. (2) Jin, W. J.; Ferna´ndez-Argu ¨ elles, M. T.; Costa-Ferna´ndez, J. M.; Pereiro, R.; Sanz-Medel, A. Chem. Commun. 2005, 883-885. (3) Ferna´ndez-Argu ¨ elles, M. T.; Jin, W. J.; Costa-Ferna´ndez, J. M.; Pereiro, R.; Sanz-Medel, A. Anal. Chim. Acta 2005, 549, 20–25. (4) Li, H. B.; Zhang, Y.; Wang, X. Q.; Xiong, D. J.; Bai, Y. Q. Mater. Lett. 2007, 61, 1474–1477. (5) Wang, L. Y.; Wang, L.; Gao, F.; Yu, Z. Y.; Wu, Z. M. Analyst 2002, 127, 977–980. (6) Chen, X. D.; Dong, Y. P.; Fan, L.; Yang, D. C. Anal. Chim. Acta 2007, 582, 281–287. (7) Chen, H. Q.; Wang, L.; Liu, Y.; Wu, W. L.; Liang, A. N.; Zhang, X. L. Anal. Bioanal. Chem. 2006, 385, 1457–1461. (8) Yao, H. Q.; Zhang, Y.; Xiao, F.; Xia, Z. Y.; Rao, J. H. Angew. Chem., Int. Ed. 2007, 46, 4346–4349. (9) Huang, C. P.; Li, Y. K.; Chen, T. M Biosens. Bioelectron. 2007, 22, 1835– 1838. (10) Liang, J. G.; Huang, S.; Zeng, D. Y.; He, Z. K.; Ji, X. H.; Ai, X. P.; Yang, H. X. Talanta 2006, 69, 126–130. (11) Cordes, D. B.; Gamsey, S.; Singaram, B. Angew. Chem., Int. Ed. 2006, 45, 3829–3832. (12) Traviesa-Alvarez, J. M.; Sa´nchez-Barraga´n, I.; Costa-Ferna´ndez, J. M.; Pereiro, R.; Sanz-Medel, A. Analyst 2007, 132, 218–223. (13) de Mello Donega´, C.; Bol, A. A.; Meijerink, A. J. Lumin. 2002, 96, 87–93. (14) Chung, J. H.; Ah, C. S.; Jang, D. J. J. Phys. Chem. B 2001, 105, 4128–4132. (15) Cheng, B. C.; Wang, Z. G. Adv. Funct. Mater. 2005, 15, 1883–1890. 10.1021/ac800100y CCC: $40.75  2008 American Chemical Society Published on Web 04/12/2008

phosphor powders for color displays.16–18 Nevertheless, QDs have seldom been explored for RTP detection in aqueous solution.19 Herein, we report Mn-doped ZnS QDs for the RTP detection of enoxacin in biological fluids. Enoxacin is chosen as the target as it is one kind of quinolones, an important group of antibiotics widely used as human and veterinary medicine for the treatment of pulmonary, urinary, and digestive infections. Public and scientific concerns about the potential risk of quinolones to human health have been increasing due to their latent allergic hypersensitivity reactions or toxic effects on the articular cartilages causing arthralgia or juvenile arthropaties.20 Therefore, the development of new methods for detecting quinolones has captured great interest.21–29 Spectrofluorometric method is rapid, facile, and inexpensive, so it has been widely used in the determination of quinolones.25–29 However, traditional fluorescence method suffers from the interference of nonspecific fluorescence. In this report, the phosphorescence property of Mn-doped ZnS QDs is investigated to establish a RTP method for the rapid, facile, selective, and inexpensive detection of enoxacin in biological fluids to overcome the problems with nonspecific fluorescence interference. Conventional RTP detection is usually performed onto solid supports (e.g., filter paper or polymeric resin) or in liquid state in the presence of deoxidants and other inducers.30 However, the Mn-doped ZnS QDs-based RTP method reported here does not need the use of deoxidants and other inducers and allows the detection of enoxacin in biological fluids without interference from autofluorescence and the scattering light of the matrix. EXPERIMENTAL SECTION Reagents. All reagents used were of analytical grade. ZnSO4 · 7H2O, MnCl2 · 4H2O, Na2S · 9H2O, L-cysteine, enoxacin, and tris(hydroxymethyl)aminomethane (Tris) were purchased from Tianjin Kaitong Chemicals Co. (Tianjin, China), the Second Chemicals Co. of Shengyang (Shenyang, China), Tianjin Sitong Chemicals Co. (Tianjin, China), Tianjin Guangfu Chemicals Co. (Tianjin, China), Wuhan Yuanda Chemicals Co. (Wuhan, China), and Beijing Aoboxin Biotechnology, Ltd. Co. (Beijing, China), respectively. A Tris-HCl buffer solution (0.01 mol L-1 of Tris, pH 7.4) was used in the experiments. Doubly deionized water (18.2 (16) Ozawa, L.; Makimura, M.; Itoh, M. Mater. Chem. Phys. 2005, 93, 481– 486. (17) Manoharan, S. S.; Goyal, S.; Rao, M. L.; Nair, M. S.; Pradhan, A. Mater. Res. Bull. 2001, 36, 1039–1047. (18) Chen, S. H.; Greeff, A. P.; Swart, H. C. J. Lumin. 2005, 113, 191–198. (19) Thakar, R.; Chen, Y. C.; Snee, P. T. Nano Lett. 2007, 7, 3429–3432. (20) Botsoglou, N. A.; Fletouris, D. J. Handbook of Food Analysis; Nollet, L. M. L., Ed.; Marcel Dekker: Ghent, Belgium, 2004; pp 931-1037. (21) Ye, Z. Q.; Weinberg, H. S.; Meyer, M. T Anal. Chem. 2007, 79, 1135– 1144. (22) Golet, E. M.; Alder, A. C.; Hartmann, A.; Ternes, T. A.; Giger, W. Anal. Chem. 2001, 73, 3632–3638. (23) Turiel, E.; Bordin, G.; Rodrı´guez, A. R. J. Chromatogr., A 2003, 1008, 145– 155. (24) Juan-Garcı´a, A.; Font, G.; Pico´, Y. Electrophoresis 2006, 27, 2240–2249. (25) Ocan ˜a, J. A.; Callejo´n, M.; Barraga´n, F. J. Analyst 2000, 125, 1851–1854. (26) Ocan ˜a, J. A.; Barraga´n, F. J.; Callejo´n, M. Analyst 2000, 125, 2322–2325. (27) Ocan ˜a, J. A.; Barraga´n, F. J.; Callejo´n, M. Talanta 2004, 63, 691–697. (28) Xiao, Y.; Wang, J. W.; Feng, X. G.; Wang, H. Y. J. Anal. Chem. 2007, 62, 438–443. (29) Vı´lchez, J. L.; Taoufiki, J.; Ballesteros, O.; Navalo´n, A. Microchim. Acta 2005, 150, 247–252. (30) Kuijt, J.; Ariese, F.; Brinkman, U. A.; Gooijer, C. Anal. Chim. Acta 2003, 488, 135–171.

MΩ cm-1) was obtained from a Water Pro water purification system (Labconco Corporation, Kansas City, MO). Apparatus. The morphology and microstructure of the QDs were characterized by high resolution transmission electron microscopy (HRTEM) on a Philips Tecnai G2 F20 (Philips, Holland) microscope operating at a 200 kV accelerating voltage. The samples for TEM were obtained by drying sample droplets from water dispersion onto a 300-mesh Cu grid coated with a lacey carbon film, which was then allowed to dry prior to imaging. The X-ray diffraction (XRD) spectra were collected on a Rigaku D/max2500 X-ray diffractometer (Rigaku, Japan) with Cu KR radiation. The FT-Raman spectra were recorded with a RFS 100 FI-Raman spectrometer (Bruker, Germany) equipped with a 1064 nm Nd:YAG laser system with a maximum power out of 2 W, a quartz beamsplitter, and a cryogenically cooled Ge detector. The FT-IR spectra (4000-400 cm-1) in KBr were recorded using a Magna560 spectrometer (Nicolet, Madison, WI). The mixed solution of enoxacin and Mn-doped ZnS QDs was condensed and dried for detecting their interactions by FT-IR. The phosphorescence measurements were performed on an F-4500 spectrofluorometer with excitation wavelength at 316 nm (Hitachi, Japan) equipped with a plotter unit and a quartz cell (1 cm × 1 cm) in the phosphorescence mode. The experimental conditions of the F-4500 spectrofluorometer used are summarized in the Supporting Information (SI), Table S-1. Synthesis of the Mn-Doped ZnS QDs.31 To a three-necked flask, 50 mL of 0.02 M L-cysteine, 5 mL of 0.1 M ZnSO4, and 1.5 mL of 0.01 M MnCl2 were added. The mixed solution was adjusted to pH 11 with 1 M NaOH and stirred under argon at room temperature for 30 min. Then 5 mL of 0.1 M Na2S was quickly injected into the solution. The mixture was stirred for 20 min, and then the solution was aged at 50 °C under air for 2 h to form L-cysteine capped Mn-doped ZnS QDs. The ZnS QDs were synthesized using a similar procedure but without the addition of 1.5 mL of 0.01 M MnCl2. These QDs were precipitated with ethanol, separated by centrifuging, washed with ethanol, and dried in a vacuum. The prepared QDs powder is highly soluble in water. Measurement Procedure. The phosphorescence measurements were carried out with the excitation wavelength of 316 nm in the absence or presence of a series of enoxacin solution when the spectrophotometer was set in the phosphorescence mode. The slit widths of excitation and emission were 10 and 20 nm, respectively. To a 10 mL calibrated test tube 1 mL of 50 mg L-1 Mn-doped ZnS QDs solution, 1.0 mL of Tris-HCl (pH 7.4), and a given concentration of enoxacin standard solution or aqueous sample solution were sequentially added. The mixture was then diluted to volume with high purity water and mixed thoroughly. Urine and Serum Samples. The urine and serum samples were collected from healthy volunteers. The urine samples were collected at a certain period over 8 h to monitor the timedependent concentration of enoxacin in the urine after the oral administration of 400 mg of enoxacin medication. An appropriate (50-fold) dilution of urine and serum was made before detection. No further complex pretreatment and deproteinization procedures were needed in the sample preparation. (31) Zhuang, J. Q.; Zhang, X. D.; Wang, G.; Li, D. M.; Yang, W. S.; Li, T. J. J. Mater. Chem. 2003, 13, 1853–1857.

Analytical Chemistry, Vol. 80, No. 10, May 15, 2008

3833

Figure 2. RTP spectra of 5 mg L-1 aqueous solution of Mn-doped ZnS QDs aged at 50 °C for 2 h (curve 1) and at 100 °C for 2 h (curve 2) and 5 mg L-1 aqueous solution of ZnS QDs (curve 3). The inset shows the decay curve of phosphorescence emission of 5 mg L-1 aqueous solution of Mn-doped ZnS QDs aged at 50 °C for 2 h.

Figure 1. (a) HRTEM image of Mn-doped ZnS QDs; (b) Raman spectra of L-cysteine and L-cysteine capped Mn-doped ZnS QDs; and (c) XRD patterns of Mn-doped ZnS QDs aged at 50 °C for 2 h (curve 1) and aged at 100 °C for 2 h (curve 2).

RESULTS AND DISCUSSION Preparation and Characterization of the Mn-Doped ZnS QDs. The aqueous synthesis of ZnS QDs and Mn-doped ZnS QDs were carried out on the basis of the reaction of zinc sulfate, manganese chloride, and sodium sulfide according to a previously reported procedure.31 In a typical preparation of Mn-doped ZnS QDs, an aqueous mixture of 50 mL of 0.02 M L-cysteine, 5 mL of 0.1 M ZnSO4, and 1.5 mL of 0.01 M MnCl2 at pH 11 was stirred under argon at room temperature for 30 min. Then 5 mL of 0.1 M Na2S was quickly injected into the solution and stirred for 20 min. The reaction solution was aged at 50 °C under air for 2 h to form L-cysteine capped Mn-doped ZnS QDs, which were separated and purified by precipitating in ethanol. The QDs were dried and characterized by HRTEM (Figure 1a), FT-Raman spectroscopy (Figure 1b), and XRD spectroscopy (Figure 1c). The HRTEM image reveals Mn-doped ZnS QDs with spherical shape and almost uniform size in diameter about 3.5 nm (Figure 3834

Analytical Chemistry, Vol. 80, No. 10, May 15, 2008

1a). Owing to the covalent interaction between thiols and the surface of Mn-doped ZnS QDs, the S-H vibration (2552 cm-1) disappeared whereas the characteristic peak for -CH(NH2)COOH (∼1585 cm-1) and -CH2- (∼2928 cm-1) still existed in the FTRaman spectra of L-cysteine stabilized Mn-doped ZnS QDs (Figure 1b (1) cf. Figure 1b (2)). The XRD pattern of the QDs aged at 100 °C (Figure 1c, curve 2) shows more distinguishable (111), (220), and (311) planes than that of those QDs aged at 50 °C (Figure 1c, curve 1), indicating that the more stable zinc blend formed after aging at 100 °C. Phosphorescence Characteristics of the Mn-Doped ZnS QDs. The Mn-doped ZnS QDs gave no phosphorescence before aging, but show a phosphorescence emission peak at 590 nm when excited at 316 nm after aging at 50 and 100 °C. An aging temperature of 50 °C (Figure 2, curve 1) gave nearly three times improvement in the phosphorescence intensity in comparison with an aging temperature of 100 °C (Figure 2, curve 2). Therefore, the Mn-doped ZnS QDs aged at 50 °C were employed for further experiments to get better phosphorescence intensity. The phosphorescence lifetime of 2 ms for the prepared Mndoped ZnS QDs was evaluated from the decay curve of the phosphorescence emission of Mn-doped ZnS QDs (inset of Figure 2). The observed phosphorescence can be attributed to the transition of Mn2+ 4T1-6A1.14 However, without Mn doping, the ZnS QDs show no phosphorescence peaks (Figure 2, curve 3). Feasibility of the Mn-Doped ZnS QDs for the RTP Detection of Enoxacin. To explore the potential of the Mn-doped ZnS QDs for RTP detection, we investigated the effect of enoxacin on the phosphorescence of the Mn-doped ZnS QDs. Spectroscopic measurements were performed in aqueous solution buffered with Tris-HCl solution at pH 7.4 (0.01 M Tris). Figure 3 shows the RTP quenching response of the Mn-doped ZnS QDs to enoxacin in aqueous solution. The addition of enoxacin quenched the RTP emission of Mn-doped ZnS QDs with a little blue shift. The quenched phosphorescence intensity increased as the pH value varied from 6 to 7, then leveled off from pH 7 to 7.5 (See SI, Figure S-1). Further increase in pH value decreased the quenched phosphorescence intensity (See SI, Figure S-1). The effect of pH on the determination of the analyte is related to the

Figure 3. Enoxacin concentration-dependent RTP emission of the synthesized water-soluble Mn-doped ZnS QDs.

luminescence of the Mn-doped ZnS QDs as the phosphorescence intensity of the Mn-doped ZnS QDs is significantly pH-dependent (see SI, inset of Figure S-1). It is also possible that the pH effect is associated with the interaction of enoxacin with the Mn-doped ZnS QDs by changing the speciation of enoxacin. Enoxacin can be present as three chemical species: anionic, neutral zwitterionic, and cationic forms, depending on pH.32,33 The carboxylic group of enoxacin is deprotonated at pH < 6.32 (pKa1), which benefits the interaction between enoxacin and Mn2+/Zn2+ on the surface of the QDs.32,33 However, the phosphorescence intensity of the QDs is also pH-dependent, i.e., increases a little at pH 6-7, keeps stable in the range of pH 7-8.5, and then increases abruptly from pH 8.5 to 10 (See SI, inset of Figure S-1). Thus, the quenched phosphorescence intensity decreases at pH