Determination of ciprofloxacin with functionalized cadmium sulfide nanoparticles as a fluorescence probe

Spectrochimica Acta Part A 71 (2008) 1204–1211

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa

Determination of ciprofloxacin with functionalized cadmium sulfide nanoparticles as a fluorescence probe Dan Li a,b , Zheng-Yu Yan a,b,∗ , Wei-Qing Cheng a,b a b

Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, Nanjing 210009, China Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, China

a r t i c l e

i n f o

Article history: Received 16 October 2007 Received in revised form 11 March 2008 Accepted 18 March 2008 Keywords: Cadmium sulfide nanoparticle Thioacetamide Fluorescence enhancement effect Ciprofloxacin

a b s t r a c t A novel assay of ciprofloxacin with a sensitivity at the microgram level is proposed based on the measurement of enhanced fluorescence intensity signals resulting from the interaction of functionalized nano-CdS with ciprofloxacin. The CdS nanoparticles was synthesized by thioacetamide (TAA) and cadmium nitrate (Cd(NO3 )2 ) in the alkaline solution. At pH 7.4, the fluorescence signals of functionalized nano-CdS were greatly enhanced by ciprofloxacin with the increase concentration of ciprofloxacin. Linear relationship can be established between the enhanced fluorescence intensity and ciprofloxacin concentration in the range of (1.25–8.75) × 10−4 mg mL−1 ((3.77–26.4) × 10−4 mmol L−1 ) or (8.75–1200) × 10−4 mg mL−1 ((26.4–3625) × 10−4 mmol L−1 ). The limit of detection is 7.64 × 10−6 mg mL−1 (2.31 × 10−5 mmol L−1 ). Based on this, a new direct quantitative determination method for ciprofloxacin in human serum samples without separation of foreign substances was established. The contents of ciprofloxacin in human serum samples were determined with recovery of 95–105% and relative standard deviation (R.S.D.) of 1.5–2.5%. This method was proved to be very sensitive, rapid, simple and tolerance of most interfering substances. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Quinolones are broad-spectrum antibacterial agents which mechanism of action is the inhibition of DNA gyrase and DNA topoisomerase IV enzymes that control DNA topology and are vital for bacterial replication [1–3]. Ciprofloxacin (Scheme 1) (CPLX) [1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7(piperazinyl)-quinolone-3-carboxylic acid], which was introduced in 1987 [4] is one of the third generation members of synthetic fluoroquinolone antibiotics which exhibits greater intrinsic antibacterial activity and a broader antibacterial spectrum. Recently, this synthetic antibiotic has been widely used in the treatment of urinary and respiratory tract infections with good localized action on infected sites, and also in gastrointestinal diseases. In consequence, attention has been paid to its determination in various biological fluids (blood, urine and tissues). A variety of techniques such as spectrophotometry [5,6], fluorimetry [7–9], high-performance liquid chromatography (HPLC) [10,11], and flowinjection chemiluminescence [12,13], have been proposed for the determination of CPLX. However, they all have some limitations

∗ Corresponding author at: Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, China. Tel.: +86 25 83224365; fax: +86 25 86185179. E-mail address: [email protected] (Z.-Y. Yan). 1386-1425/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2008.03.024

in terms of sensitivity, selectivity, stability and simplicity. The common spectrophotometric method is simple, but it has weak specificity, and suffers from low sensitivity and serious interference. The HPLC is sensitive, but it possesses the characteristic of complexity and costliness. Therefore, it is crucial for bioanalytical chemists to develop new assays of CPLX. Luminescent semiconductor quantum dots (QDs), also called nanocrystals (NCs), have gained increasing attention in the past decade [14–21]. In comparison with organic dyes and fluorescent proteins, QDs have high quantum yield of fluorescence, broad excitation spectrum, and narrow/symmetric emission spectrum [22–24]. In addition, QDs exhibit high photobleaching threshold and excellent photostability. As a fluorescent reagent, it has been applied in the field of high-sensitive analysis of protein and deoxyribonucleic acid (DNA) [25–27]. At the same time, Wang et al. [28] detected some inorganic cations, such as Cu(II), Hg(II), and Ag(I) by utilizing luminescent QDs based on changes in the luminescence intensity of the QDs. Ji et al. [29] and Constantine et al. [30,31] demonstrated QDs could be a promising biosensor for the detection of paraoxon. At present, the fluorospectrophotometry is a promising method of biochemical analysis. Semiconductors with a one-dimensional structure, such as nanorods and nanowires, have received considerable attention recently, owing to their special properties [32,33]. Considerable progress has been made in the synthesis of a one-dimensional semiconductor by using laser ablation [34], template [35–37],

D. Li et al. / Spectrochimica Acta Part A 71 (2008) 1204–1211

solution [38,39], and other methods [40]. However, it is still a challenge to establish a mild and efficient method to fabricate one-dimensional nanostructural materials. Microwaves are electromagnetic waves containing electric and magnetic field components. Claimed effects of microwave irradiation include thermal effects and non-thermal effect [41]. Compared with conventional heating, microwave heating possesses the advantage of highefficiency and rapid formation of nanoparticles with a narrow size distribution. With microwave irradiation of reactants in polar solvents, temperature and concentration gradients can be avoided, providing a uniform environment for the nucleation. During the process, microwave not only provided the energy for the decomposition of the complexes, but also greatly enhanced the nucleophilic attack by reactant molecule, which accelerated the nucleation rate of CdS. In this paper, we reported a new kind of nanometer-sized luminescent particles by a microwave-heating route, and was used for the quantitative determination of CPLX. These fluorescent nanoparticles were highly resistant to photobleaching and emit bright and steady fluorescence. Therefore, functionalized nanoCdS ((CdS)n–(SCH2 COOH)m) had been employed as a fluorescence probe for the determination of CPLX. The method was simple, sensitive, and showing little interference, permitting a limit of detection of 7.64 × 10−6 mg mL−1 (2.31 × 10−5 mmol L−1 ) for CPLX. Consequently, it offered foundation for the high-selective and sensitive methods to analyze organic drug molecule. 2. Experimental 2.1. Apparatus All fluorescence measurements were made with a RF-5301PC fluorescence spectrophotometer (Tokyo, Japan) equipped with a Xe lamp and a plotter unit and a 1cm quartz cell. UV–vis absorption spectroscopic was achieved using a UV-2100 spectrophotometer (Tokyo, Japan). The transmission electron microscopic (TEM) images of the nanoparticles were obtained using a H-600 transmission electron microscope (Tokyo, Japan). The colloidal solution of the nanoparticles in water was dropped onto 50-A˚ thick carboncoated copper grids with the excess solution immediately wicked away. All pH measurements were obtained using a pHS-25 pH meter (Shanghai, China). 2.2. Reagents Appropriate amount of CPLX was directly dissolved in 0.20 mol L−1 of NaCl solution to prepare stock solution at a final concentration of 5.00 × 10−3 g mL−1 and stored at 0–5 ◦ C. Stock solution of 5.00 mmol L−1 Cd(NO3 )2 and 5.00 mmol L−1 thioacetamide (TAA) were obtained by dissolving 0.07 g Cd(NO3 )2 and 0.03 g TAA in 100 mL deionized water, respectively. Mercapto-acetic acid, cadmium nitrate, and thioacetamide were purchased from China Medicine Chemical Reagent Co. (Shanghai, China), Shanghai Jinshanting Chemical Reagent Factory (Shanghai, China), and Shanghai Chemical Reagent Central Factory (Shanghai, China), respectively. Sodium chloride, sodium hydroxide, and trihydroxymethyl aminomethane were purchased from Nanjing Jiu-tai Chemical Reagent Factory (Nanjing, China), Shanghai Lin-feng Chemical Reagent Co. (Shanghai, China), and ciprofloxacin was purchased from Nanjing No. 2 Pharmaceutical Factory (Nanjing, China). All chemicals used were of analytical reagent grade without further purification. Doubly deionized water was used throughout.

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2.3. Procedure Nearly monodisperse CdS QDs were synthesized by our group according to the scheme reported by Chen et al. with minor modifications [42]. Briefly, instead of mechanical agitation, microwave irradiation was used as synthetical method. The basic CdS colloids were prepared as follows. The synthesis of the colloidal solution was carried out in a 250 mL beaker with pH electrodes, 80 mL of 5.00 mmol L−1 cadmium nitrate was firstly added into the beaker, then the pH was firstly adjusted to 2.0 using mercaptoacetic acid, and then adjusted to 7.0 using 0.10 mol L−1 NaOH. Under vigorous stirring, 80 mL of 5.00 mmol L−1 CH3 CSNH2 was dropped into the beaker slowly. Subsequently, the emerging colloidal solution was adjusted to pH 9.0. So, the CdS nanoparticles were prepared and were water-soluble, but it was not biocompatible and unable to react with the drug [43]. Therefore, we are unable to use the colloidal solution of CdS as a fluorescence probe to analyze CPLX, directly. Here, we used microwave irradiation method to modify the as-prepared CdS nanoparticles. The procedure was prepared as follows. The mixture was sealed and placed in the center of the microwave oven and was irradiated with power 30% (the microwave operating in a 30 s cycles, on for 6 s, and off for 24 s at a total power of 1000 W) for 18 min without any stirring, then the CdS colloidal solution was dried in a lyophilizer and the final powder was stored in a refrigerator. After purification by precipitation, centrifugation, and decantation, the vacuum-dried CdS QDs (more than 30 mg) were redispersed in aqueous solution and kept in the dark for future use. After all of these steps, the CdS QDs became functionalized nanoparticles, which can be used to analyze drug molecule as a fluorescence probe. At the same time, QDs solution concentrations were estimated from the absorption spectra using the molar absorptivity at the first absorption maximum for QDs of this size reported by Schmelz et al. [44]. Transmission electron microscopy was used to identify the shape and size of the synthesized nanoparticles. The functionalized nanoparticles were used to detect CPLX by simultaneously scanning the excitation and emission monochromators from 300 nm to 700 nm. The following procedure was adopted. To a 10 mL standard flask, solutions were added according to the following order: 2 mL CdS solution (2.00 × 10−3 mol L−1 , the concentration of colloids is represented by the concentration of CdS existing in molecules), a known volume of CPLX standard solution, and 2 mL of 0.08 mol L−1 Tris buffer solution. Then the solutions were diluted with 0.20 mol L−1 NaCl and mixed thoroughly. The intensity of fluorescence intensity was measured at ex = 355 nm in a 1.00 cm fluorescence quartz cell with a slit width of 3.0 nm for excitation and emission.

3. Results and discussion 3.1. TEM images of nanoparticles TEM images of CdS nanoparticles and modified CdS nanoparticles are shown in Fig. 1. The TEM image (Fig. 1a) shows that the average size of nano-CdS is about 10 ± 0.2 nm, and the average diameter of functionalized nano-CdS (i.e. modified CdS nanoparticles) (Fig. 1b) is about 30 ± 0.2 nm. In addition, transmission electron microscopy showed that the solubilization and crosslinking steps did not result in aggregation. From the TEM images, we could deduce that water-soluble mercapto-acetic acid coated onto the surfaces of the particles could stabilize the colloidal solution.

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ized nano-CdS QDs were nearly monodisperse and homogeneous. At the same time, the maximum emission wavelength shifted blue about 40 nm after mixing functionalized nano-CdS with CPLX. The results suggested that there were some interaction between functionalized nano-CdS and CPLX. Simultaneously, the intensities were significantly enhanced. These results indicated that functionalized nano-CdS could be used as a new fluorescence probe for sensitive and selective determination of CPLX. According to literatures report, there are two kinds of emission peak on fluorescence spectra: one is the emission of band edge and the other is the surface defect-related emission [45,46]. In contrast to the emission of band edge, the surface defect-related emission shows red-shift on the fluorescence spectra. In this paper, the emission spectra of the CdS QDs showed two peaks, at 480 nm and 555 nm, which were attributed to the band edge and surface defects of CdS nanoparticles, respectively. However, the surface defect-related emission was unconspicuous, thus we concluded that the synthetical CdS QDs possessed excellent luminescence capability and favourable structure.

3.3. Optimization of reaction conditions

Fig. 1. TEM images of (a) CdS QDS and (b) functionalized nano-CdS QDS.

3.2. Characterization of CdS QDs Fig. 2 rescence width of width at

shows the absorption (a) and room temperature fluo(b) spectra of CdS QDs. It could be seen that the line the fluorescence spectrum was narrow (with the full half-maximum about 30 nm), showing that functional-

Fig. 2. Absorption (a) and room temperature fluorescence (b) spectra of CdS QDs.

3.3.1. Effect of buffer system The effect of buffer system on the fluorescence intensity of the complex of CPLX with functionalized nano-CdS QDs was shown in Fig. 3. Tris–HCl solution, HAc–NaAc, and KH2 PO4 –K2 HPO4 were used as the buffer at the same pH. It could be seen that Tris–HCl solution influences the fluorescence intensity of the complex significantly. At the same time, the optimal concentration of buffer solution was examined for nano-CdS colloidal solution by varying the concentration of the Tris–HCl from 0.02 mol L−1 to 0.14 mol L−1 at pH 7.4 and the result was shown in Fig. 4. It could be seen that the concentration influenced the fluorescence intensity of the complex significantly. When the concentration was chosen to be 0.08 mol L−1 for this assay, the fluorescence intensity hit the climax and remained stable when the concentration of buffer was kept between 0.08 and 0.11 mol L−1 . And the experimental result turned to be satisfactory. Therefore, 0.08 mol L−1 Tris–HCl was adopted in experiment as buffer solution.

Fig. 3. Variation of fluorescence spectra of CdS nanoparticles in different buffer system. From top to bottom: Tris–HCl, HAc–NaAc, KH2 PO4 –K2 HPO4 .

D. Li et al. / Spectrochimica Acta Part A 71 (2008) 1204–1211

Fig. 4. Effect of buffer concentration on the fluorescence intensity. Error bars represent standard deviation of four measurements at each buffer concentration.

3.3.2. Effect of pH The effect of pH on the fluorescence intensity of the complex of CPLX with functionalized nano-CdS QDs was shown in Fig. 5. Tris–HCl solution was used as the buffer. It could be seen that pH influenced the fluorescence intensity of the complex significantly. The maximum value of (F − F0 )/F0 was obtained in the pH range of 6.0–9.0. Since the intensity at pH 7.4 was the highest and remained stable when the pH was kept in the range of 7.3–7.5, at the same time, the normal pH of blood was 7.35–7.45 in vivo. Therefore, the optimal pH was chosen to be 7.4 in this study. It is known to us that there is NH group in CPLX molecule. In the neutral, soft alkaline solution, the CPLX molecule could interact with other molecules by the electrostatic interaction formed between the negative charged carboxylate groups and NH group. This electrostatic interaction COO− · · ·H N+ H caused the rigidity of lomefloxacin molecule became stronger and the fluorescence intensity of complex to be substantially increased. But at hard acidic pH, H+ in solvent could form hydrogen bonds with the NH, carbonyl group simultaneously. Similarly in hard alkaline condition,

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Fig. 6. Effect of NaCl concentration on fluorescence intensity. Error bars represent standard deviation of four measurements at each NaCl concentration.

the carboxyl group could dissociate to be COO− , which disrupted the intermolecular hydrogen bond between the NH group and H of carboxyl group, and thus the fluorescence intensity of CPLX decreased. These results revealed that the electrostatic interaction may be an effective way to increase the emission intensity of the complexes (CdS–CPLX nanocomposites). At the same time, in the neutral, soft alkaline solution, with the release of S2− from TAA, the reaction between thioacetamide and cadmium nitrate could be promoted.

3.3.3. Effect of ionic strength The ionic strength of the aqueous medium seemed to have an unconspicuous effect on the interactions between functionalized nano-CdS QDs and CPLX. As Fig. 6 showed, the value of (F − F0 )/F0 increased slowly with increasing NaCl concentration. The value for (F − F0 )/F0 reached a maximum when the concentration of NaCl was chosen to be 0.20 mol L−1 . So the ionic strength at 0.20 mol L−1 (NaCl) was chosen to obtain strong fluorescence signals.

3.3.4. Effect of reaction time and temperature Initial experiments demonstrated that the CPLX enhancing of the nano-CdS QDs was finished within 5 min and the fluorescence signals were stable for more than 30 min. We recorded the fluorescence intensity after the system had reacted for 10 min at room temperature.

Fig. 5. Effect of pH on the fluorescence intensity. Error bars represent standard deviation of four measurements at each pH.

3.3.5. Effect of colloid concentration When the fluorescence intensity of the complex (CdS–CPLX nanocomposites) was strong, the extent of the changes in fluorescence intensity was limited as well as low sensitivity with the increasing concentration of CPLX. However, while the fluorescence signals was weak, the fluorescence intensity changed significantly, but it may result in narrow linear range. In order to discuss the results within the linear concentration range, the colloid concentration was chosen to be 2.00 × 10−3 mol L−1 (the concentration of colloids was represented by the concentration of CdS existing in molecules).

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Table 1 Tests for the interference of coexisting substancesa Coexisting substance

Coexisting concentration (mg mL−1 )

Change of fluorescence intensity (%)

Calf thymus DNA Human serum albumin Glucose Na(I) K(I) Al(III) Cd(II) Ca(II) Pb(II) Hg(II) Mn(II) Fish sperm DNA Bovine serum albumin Citric acid Ag(I) Cu(II) Mg(II) Zn(II) Ni(II) Fe(III) Fe(II) Ba(II)

10.00 15.00 20.00 6.00 5.50 3.50 1.90 1.50 0.50 4.00 3.70 10.00 15.00 20.00 0.80 0.60 0.70 0.80 4.00 3.00 3.50 5.00

2.0 3.0 1.8 −2.5 −3.1 −4.5 −8.5 −9.5 −6.0 6.5 −6.2 2.5 2.0 1.6 −7.5 −8.4 −7.5 −6.5 −2.1 −3.5 −2.8 1.5

a

Ciprofloxacin, 0.10 mg mL−1 ; functionalized CdS, 2.00 × 10−3 mol L−1 ; pH 7.4.

3.4. Interference of coexisting foreign substances Many compounds have the potential to quench QDs fluorescence emissions [47,48]. The influences of familiar foreign coexisting substances were tested. The results in Table 1 indicate that the method was free from interference from most of metal ions, nucleic acids, bovine serum albumin (BSA), human serum albumin (BSA), glucose and citric acid. Therefore, the present procedure was unaffected by the interference from human serum albumin and could be used for the direct determination of CPLX in the human body fluid samples without separating human serum albumin.

porated into one, the correlation coefficient (r) would less than 0.99. Therefore, the linear ranges should be displayed, respectively. From the dynamic ranges and detection limits for ciprofloxacin, it was very clear that this method was sensitive. Even though the same experimental procedure was employed in the procedure of the QD synthesis, the many experimental variables would mean that each batch of QDs would have different fluorescence emission/absorption profiles. Thus, for each batch of QDs, a calibration curve (fluorescence versus ciprofloxacin concentration) must be constructed. 3.6. Precision and accuracy

3.5. Calibration and sensitivity The emission spectra of CdS QDs and its fluorescence titration with CPLX were recorded in aqueous solution, the results of which were shown in Fig. 7. The fluorescence intensity in the fluorescence spectrum of the complex can be correlated with the concentration of CPLX. It was found that CPLX enhances the fluorescence of CdS–CPLX nanocomposites in a concentration dependence that is best described as follows:

To assess the precision and accuracy of the method, determinations were carried out for a set of 10 measurements of 5.15 mg mL−1 CPLX under optimal condition. The average result for 10 determi-

F = F − F0 = a + kC F and F0 are the fluorescent intensities of the CdS QDs at a given CPLX concentration and at an initial concentration, respectively. a and b are the characteristic constant, k and m are the proportionality constant comprising of the “instrumental geometry factor” and related parameters. The calibration for CPLX was constructed from the results obtained under the optimal conditions. Those equations, along with limits of detection and precision, were obtained according to the general procedure. The proportional correlation of the enhancement intensity of fluorescence with the concentration of ciprofloxacin is in the range (1.25–8.75) × 10−4 mg mL−1 ((3.77–26.4) × 10−4 mmol L−1 ) or (8.75–1200) × 10−4 mg mL−1 ((26.4–3625) × 10−4 mmol L−1 ), and the linear regression equation are as follows: F = 1.62C + 0.8913 (r = 0.9952) (Fig. 8a) or F = 0.0794C + 8.0527 (r = 0.9988) (Fig. 8b), respectively. The limit of detection (S/N = 3) is 7.64 × 10−6 mg mL−1 (2.31 × 10−5 mmol L−1 ). However, if the linear ranges were incor-

Fig. 7. Variation of fluorescence spectra of CdS nanoparticles with increasing concentration of CPLX. Ccds , 2.00 × 10−3 mol L−1 ; ex = 355 nm; pH 7.4. CCPLX : (1) 0 × 10−4 mg mL−1 , (2) 1.25 × 10−4 mg mL−1 , (3) 2.50 × 10−4 mg mL−1 , (4) 5.00 × 10−4 mg mL−1 , (5) 8.75 × 10−4 mg mL−1 , (6) 10.00 × 10−4 mg mL−1 , (7) 50.00 × 10−4 mg mL−1 , (8) 100.00 × 10−4 mg mL−1 , (9) 500.00 × 10−4 mg mL−1 , and (10) 1200 × 10−4 mg mL−1 .

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Scheme 1. Structure of ciprofloxacin.

parallel experiments were conducted, and the maximum relative standard deviation (R.S.D.) was 2.5%. The recovery of the samples was between 95 and 105%. HPLC was performed as comparative method [10]. The CPLX were analyzed by using a reverse phase column LiChrospher® 60 RP-select B (50 ␮m) (EcoCART 125-3). The mobile phase was acetic acid 5%–methanol–acetonitrile (90:5:5, v/v/v). The flow rate was 0.5 mL min−1 and the analyses were performed using UV detector at 280 nm. The analyses were performed at room temperature (50 ± 2 ◦ C). The results were listed in Table 3. Compared with the comparative data, the results were reliable and satisfactory. 3.8. Mechanism of reaction between mercapto-acetic acid capped CdS and CPLX

Fig. 8. The calibration curve of CPLX concentration dependence of the fluorescence intensity of CdS QDs in the range (1.25–8.75) × 10−4 mg mL−1 (r = 0.9952) (a) or (8.75–1200.00) × 10−4 mg mL−1 (r = 0.9988) (b). Error bars represent standard deviation of four measurements at each CPLX concentration.

nations of was 5.20 mg mL−1 with the relative standard deviation of 2.0%. These values indicated that this method had good accuracy and precision. Table 2 summarizes the detection limit and linear range with different methods for the determination of CPLX. This method was comparable with ultraviolet detection [49], diodearray detector [50] and fluorimetry detection [51]. The results demonstrate that this method offers an alternative and sensitive approach for the detection of CPLX and can be used in biological specimen analysis. 3.7. Sample determination To test the method, CPLX in human serum samples containing metal ions and many foreign coexisting substances, based on the tolerance of coexisting species, were analyzed. For each sample, six Table 2 Detection of ciprofloxacin in human serum samples with different methods Methods This method HPLC–UV CE-DAD Fluorimetry

Linear range (␮g mL−1 ) 0.12–120 0.05–0.30 0.10–1.50 0.02–6.00

Detection (␮g mL−1 )

Reference

0.007 0.04 0.05 0.016

[49] [50] [51]

Fig. 7 showed that the fluorescence intensity and peak position of nano-CdS colloidal all had changed as different concentration CPLX was added. Namely, in addition to the decrease of intrinsic nano-CdS fluorescence at 495 nm, a new peak appears and grows at 450 nm, which can be explained by formation of new compound during the reaction between nano-CdS and CPLX. We found that CPLX greatly enhanced the fluorescence intensity of the nanocomposites. Firstly, because the peak position came forth blue shift, we could conclude that there was a new compound created in the process of reaction. Secondly, since the ultraviolet absorption spectra of CdS–CPLX nanocomposites did not show shift as compared to nanoCdS, a new exciplex was formed during the interaction between nano-CdS and CPLX, and the band-gap energy of the complex more than energy gap between the highest valence band (HVB) and the lowest conduction band (LCB). Thirdly, as NaCl had insignificant effect on the system, it was deduced that the primary binding power between CPLX and nanoparticles may not be electrostatic forces. At the same time, as Li once had proved [52] that surfactants could lead to the enhancing effect of the fluorescent intensity by changing the structure of compound. We assumed that the exciplex (Fig. 9) may efficiently avoid collision among molecule because of changing the surface structure of functionalized nano-CdS. Simultaneously, it might intensify the rigidity of the fluorescence molecule, which benefited for enhancing fluorescence intensity. In order to prove this hypothesis between the functionalized nano-CdS and CPLX, we had performed some experiment as follows: first of all, because the concentration of Cd2+ exceeded that of S2− (because the educt concentration of Cd2+ more than that of S2− in aqueous medium). Therefore, the surface of the nano-CdS may absorb surplus Cd2+ . However, when some substance (such as Cl− , ethylenediamine tetraacetic acid (EDTA), eriochrome black T (EBT), etc.) contained coordinate bond with Cd2+ was added into the prior functionalized nano-CdS colloid, the fluorescence characteristics of functionalized nano-CdS did not changed. Thus the unconjugated Cd2+ could not connect with the surface of functionalized nano-CdS.

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Table 3 Analytical results for human serum samplesa Human serum samples

Content of metal ions −1

HPLC method (mg mL 1 2 3 4 5 6

3.00 5.00 7.50 15.00 18.00 20.50 a

)

This method n = 6 (mg mL

−1

Recovery (%) (n = 6)

R.S.D. (%)

96–103 96–101 96–102 99–101 95–102 97–105

2.2 2.0 2.5 1.5 1.2 1.8

)

3.15 5.20 7.20 15.15 18.50 20.60

Functionalized CdS, 2.00 × 10−3 mol L−1 ; pH 7.4.

Fig. 9. A possible mechanism on interaction between CdS nanoparticles and CPLX.

At the same time, the no hydrolyzed TAA may coordinate with the Cd2+ on the surface of nano-CdS due to TAA as the reaction reagent. Consequently, the CPLX can enhanced the fluorescence intensity of nano-CdS because of the amido of the CPLX, which reacted with surface functional group of the nano-CdS. Therefore, the surface defect of nano-CdS colloid was decreased conspicuously, which could enhance the fluorescence intensity and result in red-shift on the fluorescence spectra of CdS QDs. 4. Conclusion Based on the fact that the fluorescence intensity of nanoCdS colloid greatly enhanced by CPLX, a novel method for the determination of CPLX has been proposed. It was sensitive, simple, and capable of avoiding the use of toxic dyes. In the optimum conditions, calibration graph was linear in the range (1.25–8.75) × 10−4 mg mL−1 ((3.77–26.4) × 10−4 mmol L−1 ) or (8.75–1200) × 10−4 mg mL−1 ((26.4–3625) × 10−4 mmol L−1 ). The correlation coefficient is 0.9952 or 0.9988. The limit of detection (S/N = 3) is 7.64 × 10−6 mg mL−1 (2.31 × 10−5 mmol L−1 ). The present method was applied to determine CPLX in human serum samples and the results agreed with the claimed values. The possible enhancing mechanism is due to the formation of exciplex during reaction between nano-CdS and CPLX. The new drug assay with the advantages of simplicity, rapidity, and sensitivity described here is

based on the interaction of CPLX and the nano-CdS. It is well known that the semiconductor nanoparticles have excellent luminescence properties, at the same time; organic monomers have abundant kinds and different functional groups (such as COOH, OH, NH2 , SH, etc.). So, we can use the semiconductor nanoparticles as the core and functional monomer (such as mercapto-acetic acid) as the shell to prepare the functionalized nanoparticles. On the one hand, the composite nanoparticles have excellent luminescence properties; on the other hand, its surface is covered with functional groups, thus the groups cannot only solve the largest limiting factor about the stability and solubility of nanoparticles in aqueous solutions but also combine to organic molecules. Further studies in this field will open up the way to applications of nanomaterials in analytical chemistry and analytical biochemistry. Acknowledgements This work was supported by distinguished teachers of Department of Analytical Chemistry of China Pharmaceutical University. We also thank the editors and five co-workers for help and several constructive suggestions. References [1] J.S. Chapman, N.H. Georgopapadakou, Agents Chemother. 32 (1988) 438–442.

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