Research article Received: 18 September 2014,
Revised: 1 January 2015,
Accepted: 05 January 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2871
Assay of ceftazidime and cefepime based on fluorescence quenching of carbon quantum dots Yu Huang, Ying Zhang, Zhengyu Yan* and Shenghua Liao* ABSTRACT: A novel and sensitive method for the determination of ceftazidime and cefepime in an active pharmaceutical ingredient (API) has been developed based on the fluorescence quenching of poly(ethylene glycol) (PEG)2000-capped carbon quantum dots (CQDs) prepared using a chemical oxidation method. The quenching of fluorescence intensity is proportional to the concentration of ceftazidime and cefepime over the range of 0.33–3.30 and 0.24–2.40 μg/mL, respectively. The mode of interaction between PEG2000-capped CQDs and ceftazidime/cefepime in aqueous solutions was investigated using a fluorescence, UV/Vis and Fourier transform infrared spectrometry (FTIR) at physiological pH. UV/Vis and FTIR spectra demonstrated that ground state compounds were formed through hydrophobic interaction the fluorescence quenching of CQDs caused by ceftazidime and cefepime. The quenching constants decreased with increases in temperature, which was consistent with static quenching. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: carbon quantum dots; ceftazidime; cefepime; fluorescence quenching; hydrogen bonding
Introduction Over the past two decades, quantum dots (QDs) with unique optical and electronic characteristics have attracted more and more attention. Compared with conventional organic fluorescence dyes, QDs have many advantages, such as a broaden absorption band, narrow and symmetric emission band, high fluorescence quantum yield and so on. However, most inorganic QDs (CdTe, CdSe, CdS/ZnS, etc.) consist of heavy metal ions and their further application in biology and medicine is hindered by the biological toxicity of heavy metal ions. As a novel fluorescence carbon-based nanomaterial, CQDs have some excellent properties over traditional semiconductor QDs and organic fluorescence dyes, for example, low toxicity (1–9), good biocompatibility and stable fluorescence intensity (10,11). The small molecular mass enables CQDs to easily enter the living body through endocytosis in fluorescent marking and biological imaging (12–14), and CQDs have potential in the biomedical field, especially in cell dynamic tracing and in vivo imaging (15–17). In addition, CQDs also function as sensitive probes to detect organic and biological macromolecules. Gonçalves et al. prepared CQDs via laser ablation and applied to the detection of Hg2+ (18,19). Biocompatible nanosensors of CQDs were fabricated using a hydrothermal method for the determination of Cr3+, Al3+ and Fe3+ (20). Li et al. linked CQDs with a carboxyl fluorescein (FAM)-labeled single-stranded DNA molecule through a π–π bond for the detection of DNA (21). To our knowledge, the interactions of CQDs with ceftazidime and cefepime have not been reported previously. Ceftazidime and cefepime are used to kill bacteria and inhibit other pathogenic microorganisms. Currently, the main analytical methods for dtermining ceftazidime and cefepime are microbiological assay, high-performance liquid chromatography (HPLC), UV/Vis spectrophotometry and other physical and chemical methods (22–24). Highly sensitive as these methods are, they have
Luminescence 2015
some disadbantages such as the need for complex operations and expensive equipment. Here, we fabricated CQDs by the chemical oxidation of activated carbon and investigated fluorescence quenching by ceftazidime and cefepime. The morphological and optical properties of the synthesized CQDs were assessed by transmission electron microscopy (TEM), UV/Vis spectrometer, fluorescence spectra and Fourier transform infrared spectroscopy (FTIR). This method was applied to the determination of ceftazidime and cefepime in API for the first time with satisfactory results. Under physiological pH, the method has high recovery and sensitivity, a low detection limit, wide linear range and a strong anti-interference ability. The interaction mechanisms of CQDs with ceftazidime and cefepime were explained by thermodynamic parameters and the fluorescence quenching principle. Therefore, the quenching mechanism was elucidated to be a static quenching process.
Experimental Chemicals The chemicals used in the study were as follows: analytically pure HNO3 (Nanjing Chemical Regent Co. Ltd., Nanjing, China), analytically pure poly(ethylene glycol) (PEG)2000 (Xilong Chemical Regent Co. Ltd., Shantou, China), Tris (Sinopharm Chemical Regent Co. Ltd., Shantou, China) and ceftazidime (Yangtze River
* Correspondence to: Z. Yan and S. Liao, School of Science, China Pharmaceutical University, Nanjing 210009, China. E-mail: [email protected]; [email protected] School of Science, China Pharmaceutical University, Nanjing 210009, China
Copyright © 2015 John Wiley & Sons, Ltd.
Y. Huang et al. Pharmaceutical Group Co. Ltd., Taizhou, China), cefepime [Dawnrays Pharmaceutical (Holdings) Co. Ltd., Suzhou, China]. Activated carbon was collected from smoldering sawdust and the water used was secondary ultrapure.
which is similar to those reported in literature (26–28). The fluorescence intensity of pure CQDs is very weak. By contrast, the fluorescence emission spectra of CQDs conjugated with PEG2000 exhibits a broad, strong and almost Gaussian emission band at 435 nm with the emission wavelength unchanged.
Preparation of CQDs 300
200
F /F-1= 0.024C - 0.0717
1
11
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0 350
400
450
500
550
600
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Wavelength(nm)
F /F-1 = 0.0649C - 0.1551
Reaction of CQDs with ceftazidime and cefepime
120
FL
Stock solutions (concentration, 1.0 mg/mL) were prepared by dissolving suitable amounts of ceftazidime or cefepime in water. After different volumes of ceftazidime and cefepime stock solution had been added, the fluorescence intensity of CQDs (0.1 mg/mL) in Tris/HCl solution (pH 7.40) was recorded under the following conditions: the excitation wavelength was set to 310 nm, the excitation slit width was set to 5 nm and the emission slit was set to 10 nm.
A
FL
CQDs were prepared according to our previous report (25). Typically,0.2 g of activated carbon prepared by using sawdust as carbon source was added to 50 mL of HNO3 solution (5 mol/L). The mixture was refluxed under magnetic stirring at 120°C for 8 h. The filtrate was then obtained by filtering the reaction liquid and the pH was adjusted to neutral by adding 0.1 mol/L NaOH. Unreacted activated carbon particles were removed by centrifugation. Finally, 100 mg PEG2000 was added into the above supernatant for surface modification under small fire heating in a microwave oven for 30 min. The TEM of CQDs was obtained using a transmission electron microscope ( JEM-2010 UHR). The absorption spectrum was examined on a UV2100 UV/Vis spectrometer (Shimadzu, Japan). FL spectra were measured using a RF-5301 spectrofluorophotometer (Shimadzu). All pH values were assessed with a model pH S-25 (Leici Equipment Factory, Shanghai, China). IR spectra were acquired by FTIR spectroscopy (Shimadzu FTIR-8400S).
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Results and discussion
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Wavelength(nm)
Characterization of CQDs The ultrastructure of CQDs was examined by field-emission TEM (Fig. 1A). It shows almost monodispersed and homogeneous CQDs with a diameter of 5–8 nm. The absorption spectrum and fluorescence emission spectra are given in Fig. 1(B). A broad and strong peak near 280 nm is observed in the absorption spectrum of CQDs,
Figure 2. FL spectra of CQDs upon the addition of different concentration of (A) cefepime and (B) ceftazidime in the range 0–3.30 and 2.40 μg/mL, respectively. (Inset) Plot of fluorescence intensity ratios of F0/F – 1 versus the concentration of (A) cefepime and (B) ceftazidime in Tris/HCl solution (pH 7.40) at room temperature. Error bars represent standard deviations from three measurements and relative standard deviations (RSD).
Figure 1. TEM image (A), absorption spectrum and FL spectra (B) of CQDs. (a) UV/Vis absorption spectra of CQDs. (b) FL spectra of pure CQDs. (c) FL spectra of CQDs conjugated with PEG 2000.
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Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015
Carbon quantum dots quenched by ceftazidime and cefepime Table 1. Linear regression equations for the determination of cefepime and ceftazidime in API Drug Cefepime Ceftazidime
Linear regression equationsa
Correlation coefficient (r)
Linear range (μg/mL)
Limit of detectionb (μg/mL)
0.991 0.994
0.33–3.30 0.24–2.40
5.10 × 10-3 4.70 × 10-3
F0/F – 1 = 0.012C – 0.029 F0/F – 1 = 0.033C – 0.022
a
F0 and F are the fluorescence intensities of CQDs in the absence and presence of the quencher (cefepime and ceftazidime), respectively. b Limit of detection is defined by the equation LOD = 3σ/k, where σ is the standard deviation of the measurement of blank measurements (n = 10) and k is the slope of the calibration graph.
Effect of ceftazidime and cefepime on the fluorescence spectra and UV/Vis spectra of CQDs Under optimal experimental conditions, known amounts of ceftazidime and cefepime standard solutions were added to the CQDs solution. The fluorescence intensity of CQDs could be quenched by ceftazidime and cefepime, whereas the bandwidth and maximum emission wavelength of the fluorescence spectrum of the CQDs solution remained almost unchanged (Fig. 2). Moreover, a linear relationship between the quenching fluorescence intensity of the CQDs and the concentration of antibiotics was observed (Table 1). The correlation coefficient is close to 1 and the detection limit is low. The results demonstrated that the potential applicability of a CQDs-based fluorescence probe for the detection of ceftazidime and cefepime in an active pharmaceutical ingredient (API). Reactions of CQDs with ceftazidime and cefepime were investigated under optimum conditions using UV/Vis absorption spectra. As shown in Fig. 3, when ceftazidime and cefepime were mixed with CQDs, the absorption spectra shifted from 253 to 259 nm and 256 to 254 nm, respectively. The change in the absorption spectra demonstrates that a new compound was generated (11).
Recovery
Figure 3. UV/Vis absorption spectra of cefepime (A) and ceftazidime (B). (a) UV/Vis absorption spectra of mixture of CQDs and drugs. (b) UV/Vis absorption spectra of drugs. (c) UV/Vis absorption spectra of mixture of CQDs and drugs (CQDs as the blank solution). (d) UV/Vis absorption spectra of CQDs. (Inset) Spectra amplified from the frame on the left.
To evaluate the recovery of the proposed method in API detection, three different concentrations of ceftazidime and cefepime solutions were prepared and spiked with standard solutions. The recovery and relative standard deviation (RSD) values are listed in Table 2. It can be seen that the average recovery is within the range 98–102% and the RSD is < 2.7%. Therefore, the concentrationdependent fluorescence quenching method proved to be accurate and reliable.
Table 2. Recovery (n = 6) for the determination of cefepime and ceftazidime in API Drug Cefepime
Ceftazidime
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Sample
Original found (μg/mL)
Added (μg/mL)
Found (μg/mL)
RSD% (n = 6)
Recovery (%)
1 2 3 1 2 3
3.49 6.93 9.84 3.33 6.67 9.89
3.33 3.33 3.33 3.24 3.24 3.24
6.75 10.33 13.17 6.63 9.85 13.08
2.7 1.8 2 2.5 1.9 1.5
98.0 101.3 100.0 101.7 98.3 98.2
Copyright © 2015 John Wiley & Sons, Ltd.
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Figure 4. Stern–Volmer equation at three different temperatures for cefepime (A) and ceftazidime (B) with CQDs (0.1 mg/mL). Error bars represent standard deviations from three measurements and relative standard deviations (RSD).
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Copyright © 2015 John Wiley & Sons, Ltd.
2.0957 × 1013 1.5721 × 1013 1.0526 × 1013 1.1400 × 1013 8.6000 × 1012 3.1000 × 1012 2.0957 × 105 1.5721 × 105 1.0526 × 105 1.1400 × 105 8.6000 × 104 3.1000 × 104 F0/F – 1 = 2.0957 × 105[CQ] + 0.4383 F0/F – 1 = 1.5721 × 105[CQ] + 0.1256 F0/F – 1 = 1.0526 × 105[CQ] + 0.3338 F0/F – 1 = 1.1400 × 105[CQ] + 0.0310 F0/F – 1 = 8.6000 × 104[CQ] + 0.0420 F0/F – 1 = 3.1000 × 104[CQ] + 0.0880 Ceftazidime
296 306 316 296 306 316
Cefepime and ceftazidime at the same concentration of 1 mg/mL.
b
Cefepime
a
7.40
–1.3a/–2.6b 1.3/–2.7 0.91/–1.1 0.76/–0.3 1.4/0.3 –3.1/–3.6 –3.2/–1.4 0.83/–1.7 1.0/–0.6 15.3/20.7 –0.2/–2.0 –2.5/–3.0
Quenching constant (L/mol)
70 70 60 50 70 70 80 90 70 20 60 60
Linear equation
Relative error (%)
Temperature (K)
Al3+ (Cl–) Ca2+ (Cl–) Cd2+ (Cl–) Co2+ (Cl–) Cu2+ (SO2– 4 ) Br- (K+) Cl– (K+) I– (K+) NO3– (K+) Mg2+ (SO42–) + SO2– 4 (Na ) Na+ (HCO3–)
Coexisting substance concentration(10-4 mol/L)
Drug
Coexisting substance
Table 4. Linear equations and related parameters of fluorescence quenching by cefepime and ceftazidime at different temperatures
Table 3. Interference of coexisting substances
pH
Many compounds have known effects on the fluorescence intensity of QDs (29,30). To assess the selectivity of the proposed method, the interference from some ions was tested under optimum conditions (Table 3). It was found that most ions show little interference on the determination of antibiotics, with the exception of Mg(II). Because the API was of high purity, metal ions and other additives contained in the API were limited, and the impact of Mg(II) ions and other additives could be neglected. Accordingly,
Quenching rate constant L/mol/s
Related parameters R2
Impact of coexisting substances
0.9972 0.9935 0.9813 0.9957 0.9980 0.9878
Y. Huang et al.
Luminescence 2015
Carbon quantum dots quenched by ceftazidime and cefepime the method displayed good selectivity for the determination of ceftazidime and cefepime in an API.
Quenching mechanism Temperature effect. The mechanisms of fluorescence quenching are usually classified as dynamic quenching and
A
120 100
T/%
80 a
60
static quenching (31). The quenching constant for dynamic quenching increases as the temperature increases. The relationship between the quenched fluorescence intensity of QDs and the concentration of quencher can be described using the Stern–Volmer equation (32). However, the quenching constant of static quenching increases as the temperature decreases. The relationship between fluorescence quenching intensity and the concentration of quencher can be fitted by a double reciprocal equation (33). To confirm the type of fluorescence quenching, the effect of temperature on the quenching constant was investigated. The linear relationship between the fluorescence quenching of CQDs and concentration of cefepime and ceftazidime at 296, 306 and 316 K is shown in Fig. 4 and Table 4. The quenching constants decreased with increasing temperature, which indicated a static quenching process.
b
40 c
20 4000
3500
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2500
2000
1500
1000
500
Wavenumber/cm-1
B
120 100
T/%
80
a 60
c
40 20 0 4000
FTIR spectrometry. FTIR spectrometry of CQDs, ceftazidime and cefepime, as well as CQDs in the presence of ceftazidime and cefepime, was carried out to further confirm the strategy for the detection (Fig. 5). It can be speculated that peaks on curve b at 3417 and 3194 cm-1 (Fig. 5A), 3410 and 3311 cm-1 (Fig. 5B) resulted from the stretching vibration of the amino groups of cefepime and ceftazidime, respectively. However, there is only one peak at 3430 cm–1 in the spectra of the mixture (curve a in Fig. 5A and B). A possible explanation for this is that the negative hydroxy groups on the surface of CQDs interact with the amino groups in cefepime and ceftazidime via hydrogen bonding, which leads to fluorescence quenching. Hydrogen bonding also affects the vibration frequency and electron density, which leads to a decrease in the absorption intensity in the fingerprinting area.
b
3500
3000
2500
2000
1500
1000
500
Wavenumber/cm-1 Figure 5. Fourier transform infrared spectrometry of cefepime (A) and ceftazidime (B). (a) Fourier transform infrared spectrometry of mixture of CQDs and drugs. (b) Fourier transform infrared spectrometry of drugs. (c) Fourier transform infrared spectrometry of CQDs.
The strategy for the detection of cefepime and ceftazidime. As illustrated in Fig. 6, the fluorescence intensity of CQDs is rather strong in the absence of cefepime and ceftazidime. Upon addition of the antibiotics, the hydroxy group on the surface of CQDs interacts with the amino group in cefepime and ceftazidime via hydrogen bonding, which resulted in fluorescence quenching.
Figure 6. Schematic illustration of the drugs detection based on CQDs fluorescence quenching.
Luminescence 2015
Copyright © 2015 John Wiley & Sons, Ltd.
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Y. Huang et al.
Conclusion CQDs were used for the determination of cefepime and ceftazidime in an API based on fluorescence quenching and the interaction mechanism was investigated. The results indicated that a ground state complex was formed during the interaction by hydrogen bonding. The quenching was found to be static due to the decrease in the quenching constant with increasing temperature. Acknowledgements The financial support from Scientific Innovation Research of College Graduate in Jiangsu Province (CXLX13_312) and Priority Academic Program Development of Jiangsu Higher Education Institutions are highly acknowledged.
References 1. Mao XJ, Zheng HZ, Long YJ, Du J, Hao JY, Wang LL, et al. Study on the fluorescence characteristics of carbon dots. Spectrochim Acta A 2010; 75: 553–7. 2. Huang HQ, Zeng P, Han BF Xu SK. Preparation of fluorescent carbon dots and its cytotoxicity for Saccharomyces cerevisiae. Chinese J Inorg Chem 2012; 1: 13–19. 3. Yang ST, Wang X, Wang H, Lu F, Luo PG, Cao L, et al. Carbon dots as nontoxic and high performance fluorescence imaging agents. J Phys Chem C 2009; 113: 18110–14. 4. Liu CJ, Zhang P, Zhai XY, Tian F, Li WC, Yang JH, et al. Nano-carrier for gene delivery and bioimaging based on carbon dots with PEIpassivation enhanced fluorescence. Biomaterials 2012; 33: 3604–13. 5. Ray SC, Saha A, Jana NR Sarkar R. Fluorescent carbon nanoparticles: synthesis, characterization and bioimaging application. J Phys Chem C 2009; 113: 18546–51. 6. Dong YQ, Wang RX, Li H, Shao JW, Chi YW, Lin XM, et al. Polyaminefunctionalized carbon quantum dots for chemical sensing. Carbon 2012; 50: 2810–15. 7. Lin PP, Chen JW, Chang LW, Wu JP, Redding L, Chang H, et al. Computational and ultrastructural toxicology of a nanoparticle, quantum dot 705, in mice. Environ Sci Technol 2008; 42: 6264–70. 8. Geys J, Nemmar A, Verbeken E, Smolders E, Ratoi M, Hoylaerts MF, et al. Acute toxicity and prothrombotic effects of quantum dots: impact of surface charge. Environ Health Perspect 2008; 116: 1607–13. 9. Hardman R. A toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors. Environ Health Perspect 2006; 114: 165–72. 10. Peng H, Travas-Sejdie J. Simple aqueous solution route to luminescent carbogenic dots from carbohydrates. Chem Mater 2009; 21: 5563–5. 11. Sun YP, Zhou B, Lin Y, Wang W, Fernando KAS, Pathak P, et al. Quantum-sized carbon dots for bright and colorful photoluminescence. J Am Chem Soc 2006; 128: 7756–7. 12. Wang QL, Zheng HZ, Long YJ, Zhang LY, Gao M Bai WJ. Microwave- hydrothermal synthesis of fluorescent carbon dots from graphite oxide. Carbon 2011; 49: 3134–40.
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13. Zhou JJ, Sheng ZH, Han HY, Zou MQ Li CX. Facile synthesis of fluorescent carbon dots using watermelon peel as a carbon source. Mater Lett 2012; 66: 222–4. 14. Cao L, Wang X, Meziani MJ, Lu FS, Wang HF, Luo PJG, et al. Carbon dots for multiphoton bioimaging. J Am Chem Soc 2007; 129: 11318–19. 15. Sun YP, Wang X, Lu FS, Cao L, Meziani MJ, Luo PJG, et al. Doped carbon nanoparticles as a new platform for highly photoluminescent dots. J Phys Chem C 2008; 112: 18295–8. 16. Zhao QL, Zhang ZL, Huang BH, Peng J, Zhang M Pang DW. Facile preparation of low cytotoxicity fluorescent carbon nanocrystals by electrooxidation of graphite. Chem Commun 2008; 41: 5116–18. 17. Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Ipe BI, et al. Renal clearance of quantum dots. Nat Biotechnol 2007; 25: 1165–70. 18. Gonçalves HMR, Duarte AJ, Davis F, Higson SPJ Silva JCGED. Layer-bylayer immobilization of carbon dots fluorescent nanomaterials on single optical fiber. Anal Chim Acta 2012; 735: 90–5. 19. Gonçalves H, Jorge PAS, Fernandes JRA Silva JCGED. Hg(II) sensing based on functionalized carbon dots obtained by direct laser ablation. Sens Actuators B Chem 2010; 145: 702–7. 20. Liu LQ, Li YF, Zhan L, Liu Y Huang CZ. One-step synthesis of fluorescent hydroxyls-coated carbon dots with hydrothermal reaction and its application to optical sensing of metal ions. Sci China Chem 2011; 54: 1342–7. 21. Li H, Zhang Y, Wang L, Tian JQ Sun XP. Nucleic acid detection using carbon nanoparticles as a fluorescent sensing platform. Chem Commun 2011; 47: 961–3. 22. Hu CQ. Basic theory of antibiotics quality analysis by HPLC method. Beijing: Meteor Press, 2010: 3–4. 23. Chen NJ, Zhang H. HPLC-ELSD method for the determination of roxithromycin. J Chinese Antibiot 2009; 4: 288–90. 24. State Pharmacopoeia Commission. Chinese pharmacopoeia. Beijing: Chem Ind Press, 2005: Appendix 82. 25. Lv H, Shan XH, Chen J, Jiang YH, Chen JQ Yan ZY. Preparation of carbon quantum dots in wood charcoal and their interaction with bovine serum albumin. New Carbon Mater 2013; 28: 307–12. 26. Liu CJ, Zhang P, Zhai XY, Tian F, Li WC, Yang JH, et al. Nano-carrier for gene delivery and bioimaging based on carbon dots with PEIpassivation enhanced fluorescence. Biomaterials 2012; 33: 3604–13. 27. Zhu H, Wang XL, Li YL, Wang ZJ, Yang F Yang XR. Microwave synthesis of fluorescent carbon nanoparticles with electrochemiluminescence properties. Chem Commun 2009; 34: 5118–20. 28. Hu SL, Niu KY, Sun J, Yang J, Zhao NQ Du XW. One-step synthesis of fluorescent carbon nanoparticles by laser irradiation. J Mater Chem 2009; 19: 484–8. 29. Ganjali MR, Hosseini M, Aboufazeli F, Faridbod F, Goldooz H Badiei AR. A highly selective fluorescent probe for pyrophosphate detection in aqueous solutions. Luminescence 2012; 27: 20–3. 30. Wu DD, Chen Z. ZnS quantum dots-based fluorescence spectroscopic technique for the detection of quercetin. Luminescence 2014; 29: 307–13. 31. Mátyus L, Szöllösi J Jenei A. Steady-state fluorescence quenching applications for studying protein structure and dynamics. Photochem Photobiol B 2006; 83: 223–36. 32. Chen GZ, Huang XZ Zhen ZZ. Fluorescence analytical method. Beijing: Sci Press 1990; 201: . 33. Luo KL, Zhang Q Ren LY. Fluorescence study on the interaction of nano-TiO2 and BSA. Anqing Teachers Coll (Nat Sci Edit) 2009; 15: 55–7.
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015
Revised: 1 January 2015,
Accepted: 05 January 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2871
Assay of ceftazidime and cefepime based on fluorescence quenching of carbon quantum dots Yu Huang, Ying Zhang, Zhengyu Yan* and Shenghua Liao* ABSTRACT: A novel and sensitive method for the determination of ceftazidime and cefepime in an active pharmaceutical ingredient (API) has been developed based on the fluorescence quenching of poly(ethylene glycol) (PEG)2000-capped carbon quantum dots (CQDs) prepared using a chemical oxidation method. The quenching of fluorescence intensity is proportional to the concentration of ceftazidime and cefepime over the range of 0.33–3.30 and 0.24–2.40 μg/mL, respectively. The mode of interaction between PEG2000-capped CQDs and ceftazidime/cefepime in aqueous solutions was investigated using a fluorescence, UV/Vis and Fourier transform infrared spectrometry (FTIR) at physiological pH. UV/Vis and FTIR spectra demonstrated that ground state compounds were formed through hydrophobic interaction the fluorescence quenching of CQDs caused by ceftazidime and cefepime. The quenching constants decreased with increases in temperature, which was consistent with static quenching. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: carbon quantum dots; ceftazidime; cefepime; fluorescence quenching; hydrogen bonding
Introduction Over the past two decades, quantum dots (QDs) with unique optical and electronic characteristics have attracted more and more attention. Compared with conventional organic fluorescence dyes, QDs have many advantages, such as a broaden absorption band, narrow and symmetric emission band, high fluorescence quantum yield and so on. However, most inorganic QDs (CdTe, CdSe, CdS/ZnS, etc.) consist of heavy metal ions and their further application in biology and medicine is hindered by the biological toxicity of heavy metal ions. As a novel fluorescence carbon-based nanomaterial, CQDs have some excellent properties over traditional semiconductor QDs and organic fluorescence dyes, for example, low toxicity (1–9), good biocompatibility and stable fluorescence intensity (10,11). The small molecular mass enables CQDs to easily enter the living body through endocytosis in fluorescent marking and biological imaging (12–14), and CQDs have potential in the biomedical field, especially in cell dynamic tracing and in vivo imaging (15–17). In addition, CQDs also function as sensitive probes to detect organic and biological macromolecules. Gonçalves et al. prepared CQDs via laser ablation and applied to the detection of Hg2+ (18,19). Biocompatible nanosensors of CQDs were fabricated using a hydrothermal method for the determination of Cr3+, Al3+ and Fe3+ (20). Li et al. linked CQDs with a carboxyl fluorescein (FAM)-labeled single-stranded DNA molecule through a π–π bond for the detection of DNA (21). To our knowledge, the interactions of CQDs with ceftazidime and cefepime have not been reported previously. Ceftazidime and cefepime are used to kill bacteria and inhibit other pathogenic microorganisms. Currently, the main analytical methods for dtermining ceftazidime and cefepime are microbiological assay, high-performance liquid chromatography (HPLC), UV/Vis spectrophotometry and other physical and chemical methods (22–24). Highly sensitive as these methods are, they have
Luminescence 2015
some disadbantages such as the need for complex operations and expensive equipment. Here, we fabricated CQDs by the chemical oxidation of activated carbon and investigated fluorescence quenching by ceftazidime and cefepime. The morphological and optical properties of the synthesized CQDs were assessed by transmission electron microscopy (TEM), UV/Vis spectrometer, fluorescence spectra and Fourier transform infrared spectroscopy (FTIR). This method was applied to the determination of ceftazidime and cefepime in API for the first time with satisfactory results. Under physiological pH, the method has high recovery and sensitivity, a low detection limit, wide linear range and a strong anti-interference ability. The interaction mechanisms of CQDs with ceftazidime and cefepime were explained by thermodynamic parameters and the fluorescence quenching principle. Therefore, the quenching mechanism was elucidated to be a static quenching process.
Experimental Chemicals The chemicals used in the study were as follows: analytically pure HNO3 (Nanjing Chemical Regent Co. Ltd., Nanjing, China), analytically pure poly(ethylene glycol) (PEG)2000 (Xilong Chemical Regent Co. Ltd., Shantou, China), Tris (Sinopharm Chemical Regent Co. Ltd., Shantou, China) and ceftazidime (Yangtze River
* Correspondence to: Z. Yan and S. Liao, School of Science, China Pharmaceutical University, Nanjing 210009, China. E-mail: [email protected]; [email protected] School of Science, China Pharmaceutical University, Nanjing 210009, China
Copyright © 2015 John Wiley & Sons, Ltd.
Y. Huang et al. Pharmaceutical Group Co. Ltd., Taizhou, China), cefepime [Dawnrays Pharmaceutical (Holdings) Co. Ltd., Suzhou, China]. Activated carbon was collected from smoldering sawdust and the water used was secondary ultrapure.
which is similar to those reported in literature (26–28). The fluorescence intensity of pure CQDs is very weak. By contrast, the fluorescence emission spectra of CQDs conjugated with PEG2000 exhibits a broad, strong and almost Gaussian emission band at 435 nm with the emission wavelength unchanged.
Preparation of CQDs 300
200
F /F-1= 0.024C - 0.0717
1
11
100
0 350
400
450
500
550
600
650
Wavelength(nm)
F /F-1 = 0.0649C - 0.1551
Reaction of CQDs with ceftazidime and cefepime
120
FL
Stock solutions (concentration, 1.0 mg/mL) were prepared by dissolving suitable amounts of ceftazidime or cefepime in water. After different volumes of ceftazidime and cefepime stock solution had been added, the fluorescence intensity of CQDs (0.1 mg/mL) in Tris/HCl solution (pH 7.40) was recorded under the following conditions: the excitation wavelength was set to 310 nm, the excitation slit width was set to 5 nm and the emission slit was set to 10 nm.
A
FL
CQDs were prepared according to our previous report (25). Typically,0.2 g of activated carbon prepared by using sawdust as carbon source was added to 50 mL of HNO3 solution (5 mol/L). The mixture was refluxed under magnetic stirring at 120°C for 8 h. The filtrate was then obtained by filtering the reaction liquid and the pH was adjusted to neutral by adding 0.1 mol/L NaOH. Unreacted activated carbon particles were removed by centrifugation. Finally, 100 mg PEG2000 was added into the above supernatant for surface modification under small fire heating in a microwave oven for 30 min. The TEM of CQDs was obtained using a transmission electron microscope ( JEM-2010 UHR). The absorption spectrum was examined on a UV2100 UV/Vis spectrometer (Shimadzu, Japan). FL spectra were measured using a RF-5301 spectrofluorophotometer (Shimadzu). All pH values were assessed with a model pH S-25 (Leici Equipment Factory, Shanghai, China). IR spectra were acquired by FTIR spectroscopy (Shimadzu FTIR-8400S).
B
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0
Results and discussion
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650
Wavelength(nm)
Characterization of CQDs The ultrastructure of CQDs was examined by field-emission TEM (Fig. 1A). It shows almost monodispersed and homogeneous CQDs with a diameter of 5–8 nm. The absorption spectrum and fluorescence emission spectra are given in Fig. 1(B). A broad and strong peak near 280 nm is observed in the absorption spectrum of CQDs,
Figure 2. FL spectra of CQDs upon the addition of different concentration of (A) cefepime and (B) ceftazidime in the range 0–3.30 and 2.40 μg/mL, respectively. (Inset) Plot of fluorescence intensity ratios of F0/F – 1 versus the concentration of (A) cefepime and (B) ceftazidime in Tris/HCl solution (pH 7.40) at room temperature. Error bars represent standard deviations from three measurements and relative standard deviations (RSD).
Figure 1. TEM image (A), absorption spectrum and FL spectra (B) of CQDs. (a) UV/Vis absorption spectra of CQDs. (b) FL spectra of pure CQDs. (c) FL spectra of CQDs conjugated with PEG 2000.
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Carbon quantum dots quenched by ceftazidime and cefepime Table 1. Linear regression equations for the determination of cefepime and ceftazidime in API Drug Cefepime Ceftazidime
Linear regression equationsa
Correlation coefficient (r)
Linear range (μg/mL)
Limit of detectionb (μg/mL)
0.991 0.994
0.33–3.30 0.24–2.40
5.10 × 10-3 4.70 × 10-3
F0/F – 1 = 0.012C – 0.029 F0/F – 1 = 0.033C – 0.022
a
F0 and F are the fluorescence intensities of CQDs in the absence and presence of the quencher (cefepime and ceftazidime), respectively. b Limit of detection is defined by the equation LOD = 3σ/k, where σ is the standard deviation of the measurement of blank measurements (n = 10) and k is the slope of the calibration graph.
Effect of ceftazidime and cefepime on the fluorescence spectra and UV/Vis spectra of CQDs Under optimal experimental conditions, known amounts of ceftazidime and cefepime standard solutions were added to the CQDs solution. The fluorescence intensity of CQDs could be quenched by ceftazidime and cefepime, whereas the bandwidth and maximum emission wavelength of the fluorescence spectrum of the CQDs solution remained almost unchanged (Fig. 2). Moreover, a linear relationship between the quenching fluorescence intensity of the CQDs and the concentration of antibiotics was observed (Table 1). The correlation coefficient is close to 1 and the detection limit is low. The results demonstrated that the potential applicability of a CQDs-based fluorescence probe for the detection of ceftazidime and cefepime in an active pharmaceutical ingredient (API). Reactions of CQDs with ceftazidime and cefepime were investigated under optimum conditions using UV/Vis absorption spectra. As shown in Fig. 3, when ceftazidime and cefepime were mixed with CQDs, the absorption spectra shifted from 253 to 259 nm and 256 to 254 nm, respectively. The change in the absorption spectra demonstrates that a new compound was generated (11).
Recovery
Figure 3. UV/Vis absorption spectra of cefepime (A) and ceftazidime (B). (a) UV/Vis absorption spectra of mixture of CQDs and drugs. (b) UV/Vis absorption spectra of drugs. (c) UV/Vis absorption spectra of mixture of CQDs and drugs (CQDs as the blank solution). (d) UV/Vis absorption spectra of CQDs. (Inset) Spectra amplified from the frame on the left.
To evaluate the recovery of the proposed method in API detection, three different concentrations of ceftazidime and cefepime solutions were prepared and spiked with standard solutions. The recovery and relative standard deviation (RSD) values are listed in Table 2. It can be seen that the average recovery is within the range 98–102% and the RSD is < 2.7%. Therefore, the concentrationdependent fluorescence quenching method proved to be accurate and reliable.
Table 2. Recovery (n = 6) for the determination of cefepime and ceftazidime in API Drug Cefepime
Ceftazidime
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Sample
Original found (μg/mL)
Added (μg/mL)
Found (μg/mL)
RSD% (n = 6)
Recovery (%)
1 2 3 1 2 3
3.49 6.93 9.84 3.33 6.67 9.89
3.33 3.33 3.33 3.24 3.24 3.24
6.75 10.33 13.17 6.63 9.85 13.08
2.7 1.8 2 2.5 1.9 1.5
98.0 101.3 100.0 101.7 98.3 98.2
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Figure 4. Stern–Volmer equation at three different temperatures for cefepime (A) and ceftazidime (B) with CQDs (0.1 mg/mL). Error bars represent standard deviations from three measurements and relative standard deviations (RSD).
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2.0957 × 1013 1.5721 × 1013 1.0526 × 1013 1.1400 × 1013 8.6000 × 1012 3.1000 × 1012 2.0957 × 105 1.5721 × 105 1.0526 × 105 1.1400 × 105 8.6000 × 104 3.1000 × 104 F0/F – 1 = 2.0957 × 105[CQ] + 0.4383 F0/F – 1 = 1.5721 × 105[CQ] + 0.1256 F0/F – 1 = 1.0526 × 105[CQ] + 0.3338 F0/F – 1 = 1.1400 × 105[CQ] + 0.0310 F0/F – 1 = 8.6000 × 104[CQ] + 0.0420 F0/F – 1 = 3.1000 × 104[CQ] + 0.0880 Ceftazidime
296 306 316 296 306 316
Cefepime and ceftazidime at the same concentration of 1 mg/mL.
b
Cefepime
a
7.40
–1.3a/–2.6b 1.3/–2.7 0.91/–1.1 0.76/–0.3 1.4/0.3 –3.1/–3.6 –3.2/–1.4 0.83/–1.7 1.0/–0.6 15.3/20.7 –0.2/–2.0 –2.5/–3.0
Quenching constant (L/mol)
70 70 60 50 70 70 80 90 70 20 60 60
Linear equation
Relative error (%)
Temperature (K)
Al3+ (Cl–) Ca2+ (Cl–) Cd2+ (Cl–) Co2+ (Cl–) Cu2+ (SO2– 4 ) Br- (K+) Cl– (K+) I– (K+) NO3– (K+) Mg2+ (SO42–) + SO2– 4 (Na ) Na+ (HCO3–)
Coexisting substance concentration(10-4 mol/L)
Drug
Coexisting substance
Table 4. Linear equations and related parameters of fluorescence quenching by cefepime and ceftazidime at different temperatures
Table 3. Interference of coexisting substances
pH
Many compounds have known effects on the fluorescence intensity of QDs (29,30). To assess the selectivity of the proposed method, the interference from some ions was tested under optimum conditions (Table 3). It was found that most ions show little interference on the determination of antibiotics, with the exception of Mg(II). Because the API was of high purity, metal ions and other additives contained in the API were limited, and the impact of Mg(II) ions and other additives could be neglected. Accordingly,
Quenching rate constant L/mol/s
Related parameters R2
Impact of coexisting substances
0.9972 0.9935 0.9813 0.9957 0.9980 0.9878
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Luminescence 2015
Carbon quantum dots quenched by ceftazidime and cefepime the method displayed good selectivity for the determination of ceftazidime and cefepime in an API.
Quenching mechanism Temperature effect. The mechanisms of fluorescence quenching are usually classified as dynamic quenching and
A
120 100
T/%
80 a
60
static quenching (31). The quenching constant for dynamic quenching increases as the temperature increases. The relationship between the quenched fluorescence intensity of QDs and the concentration of quencher can be described using the Stern–Volmer equation (32). However, the quenching constant of static quenching increases as the temperature decreases. The relationship between fluorescence quenching intensity and the concentration of quencher can be fitted by a double reciprocal equation (33). To confirm the type of fluorescence quenching, the effect of temperature on the quenching constant was investigated. The linear relationship between the fluorescence quenching of CQDs and concentration of cefepime and ceftazidime at 296, 306 and 316 K is shown in Fig. 4 and Table 4. The quenching constants decreased with increasing temperature, which indicated a static quenching process.
b
40 c
20 4000
3500
3000
2500
2000
1500
1000
500
Wavenumber/cm-1
B
120 100
T/%
80
a 60
c
40 20 0 4000
FTIR spectrometry. FTIR spectrometry of CQDs, ceftazidime and cefepime, as well as CQDs in the presence of ceftazidime and cefepime, was carried out to further confirm the strategy for the detection (Fig. 5). It can be speculated that peaks on curve b at 3417 and 3194 cm-1 (Fig. 5A), 3410 and 3311 cm-1 (Fig. 5B) resulted from the stretching vibration of the amino groups of cefepime and ceftazidime, respectively. However, there is only one peak at 3430 cm–1 in the spectra of the mixture (curve a in Fig. 5A and B). A possible explanation for this is that the negative hydroxy groups on the surface of CQDs interact with the amino groups in cefepime and ceftazidime via hydrogen bonding, which leads to fluorescence quenching. Hydrogen bonding also affects the vibration frequency and electron density, which leads to a decrease in the absorption intensity in the fingerprinting area.
b
3500
3000
2500
2000
1500
1000
500
Wavenumber/cm-1 Figure 5. Fourier transform infrared spectrometry of cefepime (A) and ceftazidime (B). (a) Fourier transform infrared spectrometry of mixture of CQDs and drugs. (b) Fourier transform infrared spectrometry of drugs. (c) Fourier transform infrared spectrometry of CQDs.
The strategy for the detection of cefepime and ceftazidime. As illustrated in Fig. 6, the fluorescence intensity of CQDs is rather strong in the absence of cefepime and ceftazidime. Upon addition of the antibiotics, the hydroxy group on the surface of CQDs interacts with the amino group in cefepime and ceftazidime via hydrogen bonding, which resulted in fluorescence quenching.
Figure 6. Schematic illustration of the drugs detection based on CQDs fluorescence quenching.
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Y. Huang et al.
Conclusion CQDs were used for the determination of cefepime and ceftazidime in an API based on fluorescence quenching and the interaction mechanism was investigated. The results indicated that a ground state complex was formed during the interaction by hydrogen bonding. The quenching was found to be static due to the decrease in the quenching constant with increasing temperature. Acknowledgements The financial support from Scientific Innovation Research of College Graduate in Jiangsu Province (CXLX13_312) and Priority Academic Program Development of Jiangsu Higher Education Institutions are highly acknowledged.
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