sensors Article
Green, Hydrothermal Synthesis of Fluorescent Carbon Nanodots from Gardenia, Enabling the Detection of Metronidazole in Pharmaceuticals and Rabbit Plasma Xiupei Yang 1, *, Mingxian Liu 1 , Yanru Yin 1 , Fenglin Tang 1 , Hua Xu 1 and Xiangjun Liao 2 1
2
*
College of Chemistry and Chemical Engineering, Chemical Synthesis and Pollution Control Key Laboratory of Sichuan Province, China West Normal University, Nanchong 637000, China;
[email protected] (M.L.);
[email protected] (Y.Y.);
[email protected] (F.T.);
[email protected] (H.X.) Exposure and Biomonitoring Division, Health Canada, 50 Colombine Driveway, Ottawa, ON K1A 0K9, Canada;
[email protected] Correspondence:
[email protected]; Tel.: +86-817-2568-081
Received: 25 February 2018; Accepted: 16 March 2018; Published: 24 March 2018
Abstract: Strong fluorescent carbon nanodots (FCNs) were synthesized with a green approach using gardenia as a carbon source through a one-step hydrothermal method. FCNs were characterized by their UV-vis absorption spectra, photoluminescence (PL), Fourier transform infrared spectroscopy (FTIR) as well as X-ray photoelectron spectroscopy (XPS). We further explored the use of as-synthesized FCNs as an effective probe for the detection of metronidazole (MNZ), which is based on MNZ-induced fluorescence quenching of FCNs. The proposed method displayed a wide linear range from 0.8 to 225.0 µM with a correlation coefficient of 0.9992 and a limit of detection as low as 279 nM. It was successfully applied to the determination of MNZ in commercial tablets and rabbit plasma with excellent sensitivity and selectivity, which indicates its potential applications in clinical analysis and biologically related studies. Keywords: fluorescent carbon nanodots; metronidazole; fluorescent quenching; biological analysis
1. Introduction Metronidazole (MNZ) is a derivative of nitroimidazole and commonly used to treat human diseases, including parasitic infection, trichomoniasis, giardiasis and amebiasis [1,2]. MNZ has also been employed as a veterinary medicine to prevent and treat infections or to promote growth and improve feed conversion efficiency [3]. However, when the accumulated dose of MNZ exceeds the therapeutic threshold for humans, some toxic effects will be caused. For instance, seizures, peripheral neuropathy and ataxia [4]. For this reason, MNZ, along with several other nitroimidazoles, has already been banned in Europe. The uncontrolled use of MNZ or accidentally distributing feeds contaminated by MNZ, may result in its residues being present in edible tissues. Therefore, it is of great importance to accurately detect the content of MNZ in pharmaceuticals and biological samples. A variety of quantitative analytical strategies has been reported for the detection of MNZ, which mainly include high performance liquid chromatography (HPLC) [5], gas chromatography (GC) [6], thin layer chromatography (TLC) [7], spectrophotometry [8] and electrochemical sensors [9–11]. Considering some drawbacks of those methods, such as the time consuming sample preparation and sophisticated instrumentation required, the need for a better analytical method remains a challenge. Apart from the aforementioned methods, fluorescence analysis has attracted considerable attention due to its relatively low cost, high sensitivity, ease of operation, reliable approach and its low detection limit [12].
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Recently, fluorescent carbon nanodots (FCNs), a new class of carbon nanomaterials, have been actively researched and well applied in biosensors, biomarkers, biomedical imaging and so on [13–17]. In striking contrast to semiconductor quantum dots, FCNs are superior fluorescent nanomaterials due to their low toxicity, high chemical stability and low environmental impact. There are many examples in the literature of using some plants [18,19], polysaccharides [20] or other organic molecules [21] for the synthesis of FCNs. In 2015, we developed a novel method for the preparation of FCNs by hydrothermal treatment of aloe. By employing the new FCNs, we successfully demonstrated a sensitive sensor for the detection of tartrazine in food samples [22]. Recently, Hatamie et al. used graphitic carbon nitride as a fluorescent carbon material for detecting MNZ [23]. Gardenia jasminoides (Ellis.) is widely cultivated in Asia, especially in the south of China. It is a genus of about 250 species of flowering plants in the family Rubiaceae, which is native to the tropical and subtropical regions of Africa, Southern Asia, Australasia and Oceania. In traditional Chinese medicine (TCM), Fructus Gardenia, the dried ripe fruit of Gardenia jasminoides (Ellis.), is used to cure patients with inflammation, viral encephalitis, hepatitis, tonsillitis, tracheitis and high fever. Here, we hope that gardenia flowers can also produce similar FCNs that can be used as fluorescent probes. To the best of our knowledge, there have been no reports of the use of gardenia as a carbon source for the synthesis of FCNs or for their use as ametronidazole fluorescent probe. In this work, we demonstrated a simple, low cost, and green preparative strategy toward water-soluble nitrogen-doped FCNs by hydrothermal treatment of gardenia at 220 ◦ C. The carbonization, surface functionalization and doping occurred simultaneously during the hydrothermal treatment, which led to the formation of the nitrogen-doped FCNs. Moreover, the fluorescence of FCNs gradually decreased with an increase in MNZ concentration. Herein, we report the development of a fluorescence sensing probe based on FCNs for the detection of trace amounts of MNZ. The analytical feature and the application of the proposed fluorescence quenching method have been fully explored. The major attributes of the proposed method are its simplicity, cost-effectiveness, convenience and environmental friendliness. 2. Experimental 2.1. Materials Metronidazole, ronidazole, secnidazole and dichloremethane were purchased from Shanghai Aladdin Co. (Shanghai, China). Sodium dihydrogen phosphate (NaH2 PO4 ·2H2 O) and disodium hydrogen phosphate dodecahydrate (Na2 HPO4 ·12H2 O) were supplied by Tianjin Fuchen Chemical Reagents Co., Ltd. (Tianjin, China). Two kinds of MNZ tablets were purchased at local drug stores. Reagents and materials, such as glucose, sucrose, starch, β-cyclodextrin, ascorbic acid, urea, NH4 Cl, NaCl, KNO3 , MgSO4 , NaCO3 and amino acids were used as received without further purification. The gardenias were obtained from the school yard of China West Normal University and washed by water for further use. The ultrapure water used throughout the experiments was purified through a UPH-II-20 Tap water purification system (Chengdu Ultrapure Technology Co., Ltd., Chengdu, China). 2.2. Apparatus The FCNs were synthesized in a 100 mL hydrothermal autoclave (Weihai Huixin Chemical Machinery Co., Ltd.). All absorption spectra were recorded on a Shimadzu UV-2550 UV-vis absorption spectrophotometer (Kyoto, Japan). Fluorescence measurements were conducted with a Cary Eclipse fluorescence spectrophotometer (Varian, Palo Alto, CA, USA). The infrared spectra were obtained on a Nicolet 6700 Fourier transform infrared (FTIR) spectrometer (Thermo Electron Corporation, Waltham, MA, USA) with passed KBr pellet at room temperature. Transmission electron microscope (TEM) measurements were carried out on a JEM-1200EX (JEOL, Tokyo, Japan) with an acceleration voltage of 250V. X-ray photoelectron spectroscopic (XPS) measurements were carried out on an ESCALAB 250Xi
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(Waltham, MA, USA) and the equipped with PF4 (peek fit 4) software was used to deconvolute the narrow scan XPS spectra. 2.3. Preparation of FCNs The FCNs were synthesized from gardenias through a simple, convenient and one-step hydrothermal method. Briefly, the fresh gardenia flowers were first washed in water and dried in sunlight, followed by oven-drying at 100 ◦ C for 10 h to allow for carbonization. Then, 2.0 g of the pretreated gardenias was added into 50 mL of H2 O. The resulting mixture was transferred into a 100-mL Teflon-lined autoclave and kept at 180 ◦ C for 6 h. After the autoclave cooled down naturally, the obtained brown-yellow solution was filtrated with a 0.22 µm membrane and the FCNs were collected by removing the unreacted organic moieties through treatment with dichloromethane. Finally, the light yellow aqueous solution containing FCNs was collected and diluted 13.3 times with ultrapure water, then stored at 4 ◦ C for further characterization and use. 2.4. Quantum Yield Measurements The quantum yield of the as-synthesized FCNs was measured on the basis of a procedure described previously. Quinine sulfate in 0.1 M H2 SO4 was used as a reference standard, for which the quantum yield was 0.54 at 360 nm reported by the literature. Absolute values of the quantum yield were calculated according to the equation Φx = Φstd
Ix Astd ηx2 , 2 A x Istd ηstd
where Φ is the quantum yield of the as-symmetrical FCNs, A is the absorbance, I is the corrected emission intensity at the excitation wavelength, and η is the refractive index of the solvent. The subscripts “std” and “x” refer to a reference standard with known quantum yield and the FCNs solution, respectively. For the sake of reducing the effects of reabsorption within the sample on the observed emission spectrum, the absorbance values (A) of all solutions in the 10 mm cuvette were always controlled under 0.1. 2.5. Preparation of Samples All tablet samples (Metronidazole Tablets, MNZ declared of 200 mg/tablet, Huanan Pharmaceutical Group Co., Ltd., Guangdong, China) were purchased in the local market. Twenty tablets were finely powdered and the equivalent of one tablet (200 mg as MNZ) was accurately weighed and extracted with 100 mL of water. It was sonicated at room temperature for 5 min. The solution was filtered through an ordinary filter paper, washed with water several times and the filtrate plus washings were diluted to the mark in a 250 mL calibrated flask. The sample solutions were diluted with water to obtain solutions where the expected concentration of MNZ was within the calibration range. An oral administration of 100 mg of MNZ was given to healthy rabbits (1.8–2.5 kg), rabbit plasma samples (2–3 mL) were collected from the veins of rabbit ears at 5.0 h after dosing. The samples were kept in an ice-bath until being centrifuged at 5000× g rpm for 15 min at 4 ◦ C. The serum was carefully collected and stored at −80 ◦ C. Before assay, 0.50 mL of serum sample was diluted with 0.50 mL of 0.60 M trichloroacetic acid aqueous solution and shaken vigorously for 15 min to deposit proteins. It was then left at 0 ◦ C for 1 h. After further centrifuging at 10,000× g rpm for 15 min, the supernatant was used as the sample in the subsequent experiments. 2.6. Detection of MNZ A stock solution of MNZ (10.0 mM) was prepared in water. Working solutions were acquired by serial dilution of the stock solutions. For the determination of MNZ, 500 µL of FCNs, 1000 µL of phosphate buffer solution (PBS, pH = 7.0) and an appropriate volume of MNZ stock solution or
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sample solution were successively added into a 5.0 mL brown centrifuge tube. The mixture was ◦ C for 10 min prior to fluorescence measurement. diluted 30 °C dilutedto to4000 4000µL μLwith withwater water and and incubated incubated at at 30 for 10 min prior to fluorescence measurement. The at 355 355 nm nm and and the the excitation excitationand andemission emission Thefluorescence fluorescencespectra spectrawere were recorded recorded under under excitation excitation at slits were set at 10 nm. The sensitivity and selectivity measurements were conducted in sextuplicate. slits were set at 10 nm. The sensitivity and selectivity measurements were conducted in sextuplicate. All Allrecoveries recoverieswere werecalculated calculatedbased basedon onthe theequation equation below: below: Recovery=(C measured − Cinitial)/Cadded, Recovery = (Cmeasured − Cinitial )/Cadded , where Cmeasured is the measured sample concentration after spiking, Cinitial is the measured sample where Cmeasured is the measured concentration after spiking, Cinitial is the measured sample background concentration and Csample added is the known concentration of the spike. background concentration and Cadded is the known concentration of the spike. 3. Results and Discussion 3. Results and Discussion 3.1. Optimization of the Synthesis 3.1. Optimization of the Synthesis To ensure excellent performance of the synthesized FCNs, optimization of parameters such as To ensure excellent performance of the synthesized FCNs, optimization of parameters such as the mass of gardenia used, the reaction time and temperature for the synthesis was undertaken and the mass of gardenia used, the reaction time and temperature for the synthesis was undertaken and the results are shown in Figures S1–S3. From Figure S1, we can see clearly that the fluorescence the results are shown in Figures S1–S3. From Figure S1, we can see clearly that the fluorescence intensity of the obtained FCNs solution reached maximum fluorescence intensity with 2.0 g gardenia intensity of the obtained FCNs solution reached maximum fluorescence intensity with 2.0 g gardenia as a carbon source and 300 μL of the final synthesized solution. As a result, 2.0 g of gardenia and as a carbon source and 300 µL of the final synthesized solution. As a result, 2.0 g of gardenia and 300 μL of the final synthesized solution were selected for the entire course of the experiment. As can 300 µL of the final synthesized solution were selected for the entire course of the experiment. As can be seen from Figure S2, the fluorescence intensity gradually increased with the reaction time up to be seen from Figure S2, the fluorescence intensity gradually increased with the reaction time up to 10 h but decreased thereafter. Therefore, 10 h was chosen as the optimal reaction time. As displayed 10inhFigure but decreased thereafter. Therefore, 10 h was chosen as optimal reaction time.was As raised displayed S3, the fluorescence intensity greatly increased as the the reaction temperature fromin Figure greatly increased as the reaction temperature was 220 raised 200 200 to S3, 220the °C.fluorescence Only a slightintensity difference in fluorescence intensity was observed between °C from and 225 ◦ C. Only a slight difference in fluorescence intensity was observed between 220 ◦ C and 225 ◦ C. to°C. 220 Hence, are action temperature of220 °C was chosen for the subsequent experiments. Hence, are action temperature of220 ◦ C was chosen for the subsequent experiments. 3.2. Characterization of FCNs 3.2. Characterization of FCNs To explore the optical properties of the FCNs, UV-vis absorption spectra and To explore the optical properties of the FCNs, UV-vis absorption spectra and photoluminescence photoluminescence (PL) were studied at room temperature. As shown in Figure 1, there is an (PL) were studied at room temperature. As shown in Figure 1, there is an absorbance band centered absorbance band centered around 298 nm in the UV-vis absorption spectrum. The peak at 298 nm around nm intothe spectrum. The peak 298 nm shows can be an ascribed to emission the π→π* can be 298 ascribed theUV-vis π→π*absorption transition of C=C [24]. The PL at spectrum optimal transition of C=C [24]. The PL spectrum shows an optimal emission peak about 431 nm when excited peak about 431 nm when excited at 355 nm. Accordingly, from the photographs shown in Figure 1 atinset, 355 nm. Accordingly, from the photographs shown in Figure 1 inset, the diluted FCNs aqueous the diluted FCNs aqueous solution is light yellow under daylight (a) but exhibits a very solution light yellow under daylight (a)abut very(b) intense blue color when irradiated with a intense is blue color when irradiated with 365exhibits nm UV alight further indicating the blue fluorescent 365 nm UV light (b) further indicating the blue fluorescent property of the FCNs. Fluorescence property of the FCNs. Fluorescence under 365 nm UV light was quenched after adding MNZ (c).under The 365 nm UV light was quenched after adding MNZ (c). The strong fluorescence of the FCNs may result strong fluorescence of the FCNs may result from the emissive traps of the nitrogen-doped surface from the emissive traps of the nitrogen-doped surface [25–27]. [25–27].
Figure1.1.UV-vis UV-visabsorption absorption(red) (red) and and fluorescence fluorescence emission emission (blue) Figure (blue) spectra spectra of of the the fluorescent fluorescentcarbon carbon nanodots (FCNs). nanodots (FCNs).
As seen with other carbon-based quantum dots [28–30], the fluorescence of the current FCNs also changes with the excitation wavelength. Figure 2A displays the maximum PL peak shifts from
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other carbon-based quantum dots [28–30], the fluorescence of the current FCNs also 5 of 15 changes with the excitation 2A displays the maximum peak 415 415 to 496 nm with awavelength. change inFigure excitation wavelength from 300PL to 420shifts nm.from Such to 415 496 nm a change excitation wavelength 300 tosurface 420from nm. Such excitation-dependent to with 496 nm with changeis in excitation wavelength 300 ofto nm. Such excitation-dependent PLinabehavior related to thefrom different states the420 nitrogen-doped PLexcitation-dependent behavior is related to the different surface states of the nitrogen-doped carbon nanodots [31]. PL behavior is related to the different surface states of the nitrogen-doped carbon nanodots [31]. After the addition of metronidazole, as shown in Figure 2B, the fluorescence After the addition of metronidazole, as shown in Figure 2B, the fluorescence intensity of the FCNs carbon nanodots [31]. After the addition of metronidazole, in Figure 2B, the fluorescence intensity of the FCNs was reduced. However, the trend of as theshown position of the fluorescence emission intensity the FCNs was reduced. the of trend of the position ofthat the fluorescence emissionof was reduced. However, the trend the position the fluorescence peak basically the peak is ofbasically the same as of inHowever, Figure 2A.The results showedemission after theisaddition peak thefluorescence same as showed in excitation-dependent Figure results showed that after the of same as is in basically Figure 2A.The results that2A.The after the addition of metronidazole, theaddition fluorescence metronidazole, the properties of FCNs did not change metronidazole, fluorescence excitation-dependent properties of FCNs did not change excitation-dependent of FCNs did not change significantly. significantly. the properties significantly.
Figure 2. Fluorescence emission spectra of FCNs obtained at different excitation wavelengths Figure 2. Fluorescence emission spectra of FCNs obtained at different excitation wavelengths without Figure Fluorescence emission spectra of (15 FCNs without2.metronidazole (A) and adding metronidazole (15 μM) at (B).different excitation wavelengths metronidazole (A) and adding metronidazole µM)obtained (B). without metronidazole (A) and adding metronidazole (15 μM) (B).
To improve the stability of FCNs for detection and analysis, the fluorescence stability of the ToTo improve the stability of detection and analysis, analysis,the the fluorescence stability of improve stability ofFCNs FCNs for for and detection and stability thethe synthesized FCNthe solution was explored the results are shown influorescence Figure 3. Even afterofbeing synthesized FCN was explored and the the results are are shown shownininFigure Figure3.3.Even Even after being synthesized FCNasolution solution explored after being irradiated with UV lampwas at 365 nm forand 2 h and results after the stability was confirmed by fluorescence irradiated with a UV lamp at 365 nm for 2 h and after the stability was confirmed by fluorescence irradiated withno a UV lamp atwas 365clearly nm forobserved 2 h and after theUV stability was confirmed by fluorescence spectroscopy, bleaching under light irradiation, indicating that FCNs spectroscopy, no bleaching was clearly observed under UV light irradiation, indicating FCNs spectroscopy, no bleaching was clearly observed under UV light irradiation, indicating that have good photo stability. Using quinine sulfate as a reference, a PL quantum yield (QY) that ofFCNs 9.82% have good sulfate as asaareference, reference,a aPL PLquantum quantum yield (QY) 9.82% have goodphoto photostability. stability.Using Using quinine quinine sulfate yield (QY) of of 9.82% was measured. was measured. was measured.
Figure 3. Photostability study of FCNs under UV light irradiation. Figure Photostability study study of Figure 3.3.Photostability of FCNs FCNsunder underUV UVlight lightirradiation. irradiation.
Figure 4 shows the typical TEM images of the as-synthesized FCN solution in the absence and Figure shows(125.00 the typical images of the solution in the absence and presence of4MNZ μM). TEM It revealed that the as-synthesized FCNs were wellFCN dispersed with regular spherical Figureof4 MNZ shows(125.00 the typical TEM images ofthe theFCNs as-synthesized FCN solution in the absence and presence μM). It revealed that were well dispersed with regular spherical shape and had an average size of 9 nm approximately. However, when metronidazole was added, presence of MNZ (125.00 µM). revealed that the FCNsHowever, were wellnanoparticles dispersed with regular spherical shape and had an average sizeItof 9 nm approximately. metronidazole was added, the morphology of the FCNs changed greatly, the size of the when became extremely shape and had an average size of 9 nm approximately. However, when metronidazole was added, the morphology of the of FCNs size and of the nanoparticles becamewas extremely irregular, the surface the changed particles greatly, became the blurred particle agglomeration clearly theirregular, morphology of the FCNs changed greatly, the size of the nanoparticles became extremely irregular, observed.the surface of the particles became blurred and particle agglomeration was clearly theobserved. surface of the particles became blurred and particle agglomeration was clearly observed.
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Figure 4. Transmission electron microscope (TEM) images of FCNs in the absence of metronidazole
Figure 4. Transmission electron microscope (TEM) in the theabsence absenceofofmetronidazole metronidazole Figure 4. Transmission electron microscope (TEM) images images of FCNs FCNs in (MNZ) (a) and presence of MNZ (b). (MNZ) (a) and presence of MNZ (b). (MNZ) (a) and presence of MNZ (b). Figure 4. Transmission electron microscope (TEM) images of FCNs in the absence of metronidazole The FTIR spectrum inofFigure shows that multiple functional groups are present on the surface (MNZ) (a) and presence MNZ 5(b).
−1 The FTIRincluding spectrum instretching Figure 5 shows that multiple functional are present on the of FCNs, the vibration characteristic absorptiongroups peaks from –OH at 3450 cmsurface The FTIR spectrum in Figure 5 shows that multiple functional groups are present on the surface −1, the asymmetric and symmetric stretching vibration peaks of COO– at 1640 cm−1 and andThe 1084 cm of FCNs, including the stretching vibration characteristic absorption peaks from –OH at 3450 FTIR spectrum in Figure 5 shows that multiple functional groups are present on the surfacecm−1−1 of FCNs, including the stretching vibration characteristic absorption peaks from –OH at 3450 cm −1 −1 1400 cm , ,respectively andof1084 cm the asymmetric and vibration symmetric stretching absorption vibration peaks COO– FCNs, the [32]. stretching characteristic peaks of from –OHatat1640 3450cm cm−1−1−and −1 including 1 and and 1084 cm , the asymmetric and symmetric stretching vibration peaks of COO– at 1640 cm −1, respectively and 1084 cm−1, the asymmetric and symmetric stretching vibration peaks of COO– at 1640 cm−1 and 1400 cm [32]. −1 respectively [32]. 1400 cm −1, respectively [32]. 1400 ,cm
Figure 5. Fourier Transform Infrared spectra of the FCNs.
To further investigate the components, surface groups and the structure of the as-synthesized Figure Fourier Transform ofof the532.04 FCNs. eV shown in the XPS FCNs, XPS was carried out.5.5. The three peaks atInfrared 284.30, spectra 398.8 and Figure Fourier Transform Infrared spectra Figure 5. Fourier Transform Infrared spectra of the the FCNs. FCNs. spectrum of these nanoparticles (Figure 6A) can be attributed to C1s, N1s, and O1s, respectively. The further investigate the components, surface groups and theofstructure of the as-synthesized XPSTo results indicate that the these nanoparticlessurface are mainly composed oxygen, well as a To further investigate components, groups and the carbon, structure of theasas-synthesized FCNs, XPS was carried out.components, The C1s three peaks at 284.30, and 532.04 eVatshown inas-synthesized the XPS To further investigate the surface groups and the structure of the limited amount of nitrogen. The spectrum (Figure 6B)398.8 shows three peaks 284.3, 285.9 FCNs, XPS was carried out. The three peaks at 284.30, 398.8 and 532.04 eV shown in and the XPS spectrum of these nanoparticles (Figure 6A) can be attributed to C1s, N1s, and O1s, respectively. The 288.1 eV, which can be assigned to C–C/C=C, C–OH/C–O–C and C=O/C=N, respectively. The FCNs, XPS was carried out. The three peaks 284.30, 398.8 and eV shown inrespectively. the XPStwo spectrum spectrum of these nanoparticles (Figure 6A)atcan be attributed to532.04 C1s, N1s, and O1s, The peaks at 398.9 and 399.8 eV in the N1s spectrum (Figure 6C) come from C–N–C and C–N groups, XPS results indicate that these nanoparticles are mainly composed of carbon, oxygen, as well as aresults of these nanoparticles (Figure 6A)nanoparticles can be attributed to C1s, composed N1s, and O1s, respectively. The XPS XPSlimited results indicate that these are mainly of carbon, oxygen, as well as a respectively. The spectrum 6D) shows two peaks 532.7 three and 531.9 eV,atwhich due and to amount ofO1s nitrogen. The(Figure C1s spectrum (Figure 6B) at shows peaks 284.3,are 285.9 indicate that these nanoparticles areC1s mainly composed of carbon, oxygen, as wellat as284.3, a limited amount limited amount of nitrogen. The spectrum (Figure 6B) shows three peaks 285.9 and the existence of can C–OH/C–O–C and bands, respectively [33,34]. results are consistent with 288.1 eV, which be assigned toC=O C–C/C=C, C–OH/C–O–C and These C=O/C=N, respectively. The two of nitrogen. The C1s spectrum (Figure 6B) shows three peaks at C=O/C=N, 284.3, 285.9 and andC–N 288.1 eV, 288.1 eV, at which can assigned to N1s C–C/C=C, C–OH/C–O–C and The which two those from FTIR. peaks 398.9 and be 399.8 eV in the spectrum (Figure 6C) come from C–N–Crespectively. groups, canpeaks berespectively. assigned to C–C/C=C, C–OH/C–O–C and C=O/C=N, respectively. The two peaks at at 398.9 and eV in the N1s 6D) spectrum 6C) comeand from C–N–C and are C–N groups, The 399.8 O1s spectrum (Figure shows (Figure two peaks at 532.7 531.9 eV, which due to 398.9 andrespectively. 399.8 eV in The the N1s spectrum(Figure (Figure 6C)shows come from C–N–C and C–N groups, respectively. The spectrum two peaks at 532.7 and 531.9 eV,consistent which are due to the existence ofO1s C–OH/C–O–C and C=O6D) bands, respectively [33,34]. These results are with those from FTIR. existence of C–OH/C–O–C C=O bands, respectively [33,34]. These results withof O1sthe spectrum (Figure 6D) showsand two peaks at 532.7 and 531.9 eV, which are dueare toconsistent the existence
those from FTIR. C–OH/C–O–C and C=O bands, respectively [33,34]. These results are consistent with those from FTIR.
Figure 6. Cont.
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Figure 6. 6. Survey SurveyX-ray X-rayphotoelectron photoelectronspectroscopy spectroscopy (XPS) spectra of FCNs (A) and high-resolution Figure (XPS) spectra of FCNs (A) and high-resolution XPS Figure 6. Survey X-ray photoelectron spectroscopy (XPS) spectra of FCNs (A) and high-resolution XPS data of C1s (B), N1s (C) and O1s (D). data of C1s (B), N1s (C) and O1s (D). XPS data of C1s (B), N1s (C) and O1s (D).
3.3. Principle of the Fluorescence Sensor 3.3.3.3. Principle of of thetheFluorescence Principle FluorescenceSensor Sensor The synthetic strategy for FCNs and the principle of MNZ sensing are represented in Scheme The synthetic strategy for FCNs the of are represented in Scheme Scheme 1. synthetic strategy for FCNsand and theprinciple principle ofMNZ MNZ sensing sensing 1. The The as-prepared FCNs exhibited strong blue fluorescence. After are therepresented addition ofin MNZ, the The1.as-prepared FCNs exhibited strong blue fluorescence. After theAfter addition MNZ, the fluorescence The as-prepared FCNs exhibited strong blue fluorescence. the of addition of MNZ, the fluorescence intensity of the FCNs decreased significantly. These results suggest that the FCNs intensity of the intensity FCNs decreased significantly. These results suggest the suggest FCNs could as a fluorescence of the FCNs decreased significantly. These that results that be theused FCNs could be used as a facile fluorescence quenching sensor for MNZ with high sensitivity based on the facile fluorescence quenching sensor for MNZ with high sensitivity based on the interaction between could be used as a facile fluorescence quenching sensor for MNZ with high sensitivity based on the interaction between MNZ and FCNs. MNZ and FCNs. interaction between MNZ and FCNs.
Scheme Schemeofofthe thesynthetic synthetic strategy strategy for for FCNs sensing. Scheme 1. 1. Scheme FCNs and andthe theprinciple principleofof ofmetronidazole metronidazole sensing. Scheme 1. Scheme of the synthetic strategy for FCNs and the principle metronidazole sensing.
MechanismofofFluorescence FluorescenceQuenching Quenching 3.4.3.4. Mechanism 3.4. Mechanism of Fluorescence Quenching Broadly speaking, various kinds of molecular interactions with the quencher molecule can Broadlyspeaking, speaking,various various kinds of molecular interactions the quencher molecule can Broadly kinds of molecular interactions with with the quencher molecule can reduce reduce the fluorescence quantum yield, such as electron or energy transfer, collisional quenching, reduce the fluorescence quantum yield, such as electron or energy transfer, collisional quenching, theexcited-state fluorescencereaction quantum yield, such as electron energy transfer, collisional quenching,are excited-state and ground-state complexorformation. The quenching mechanisms usually excited-state reaction and ground-state complex formation. The quenching are mechanisms are usually reaction and ground-state complex formation. The quenching mechanisms usually divided into divided into dynamic quenching (which results from collision) and static quenching (resulting from divided into dynamic quenching (which results from collision) and static quenching (resulting from dynamic quenching resultscomplex from collision) quenching (resulting from the formation the formation of a (which ground-state betweenand the static fluorescence material and quencher). On the theaother formation a ground-state complex between the material and quencher). Onthey the of ground-state complex thedistinguished fluorescence material andsuch quencher). the other hand, hand, of they could between be further by fluorescence features as the On relationship between other hand, they could be further distinguished by features such as the relationship between could be further by features such the relationship between viscosity, quenching anddistinguished viscosity, temperature, and as lifetime measurements. In quenching general, theand dynamic quenching and viscosity, temperature, and lifetime measurements. In general, the dynamic fluorescenceand quenching will increase with the arisedynamic in the temperature ofquenching the systemconstants due to temperature, lifetime constants measurements. In general, fluorescence fluorescence quenching constants will increase with arise in the temperature of the system due to the arise energy transfer efficiency andsystem the increase in effective times between willchanges increaseinwith in the temperature of the due to changes in thecollision energy transfer efficiency changes in the transfer efficiency and thefluorescence increase inquenching effective collisionthe times between molecules. On energy the contrary, the values of the static constants will decrease and the increase in effective collision times between molecules. On the contrary, values of the molecules. On the contrary, the values of the static fluorescence quenching constants will decrease with a rise in temperature of the system. Let us suppose that the mechanism is dynamic quenching; static fluorescence quenching constants will decrease with a rise in temperature of the system. Let us with a rise inthe temperature system.quenching; Let us suppose the mechanism is dynamic quenching; it can be described by the of Stern–Volmer equation. suppose that mechanism isthe dynamic it can that be described by the Stern–Volmer equation. it can be described by the Stern–Volmer equation. F0/F = 1 + Ksv [Q] = 1 +Kqτo[Q], F0 /F F= 1 + Ksv [Q] = 1 + Kq τo [Q], 0/F = 1 + Ksv [Q] = 1 +Kqτo[Q], where F0 and F are the FCNs fluorescence intensities at 360 nm in the absence and presence of metronidazole, respectively; SV and Kq are the Stern–Volmer quenching constant and the of where F0 and F are the FCNs K fluorescence intensities at 360 nm in the absence and presence
metronidazole, respectively; KSV and Kq are the Stern–Volmer quenching constant and the
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Sensors 2018, 18, x F are the FCNs fluorescence intensities at 360 nm in the absence and presence 8 of of 15 where F0 and metronidazole, respectively; KSV and Kq are the Stern–Volmer quenching constant and the bimolecular bimolecularconstant, quenching constant, respectively; [Q] is the concentration of metronidazole; τo is quenching respectively; [Q] is the concentration of metronidazole; and τo is theand average the average lifetime of the FCNs without any other fluorescence quencher with a general value − 8 lifetime of the FCNs without any other fluorescence quencher with a general value of 10 s. Figureof 6 10−8 s. Figure 6 shows the fluorescence of the by FCNs analyzed plotting against [Q] shows the fluorescence intensities of theintensities FCNs analyzed plotting F0 /Fby against [Q]Fat0/F293, 303, 313, at 293, 313,1 and 323 K. Table 1 summarizes SV and Kq values for each and 323 303, K. Table summarizes the calculated KSV andthe Kqcalculated values for K each temperature. As shown temperature. As shown in Figure 7, as the temperature increases, the fluorescence in Figure 7, as the temperature increases, the fluorescence quenching constant increasesquenching gradually constant increases gradually and increasing the temperature favors the fluorescence quenching. and increasing the temperature favors the fluorescence quenching. This is consistent with the This is consistent the dynamic quenching mechanism. These findings that the dynamic quenching with mechanism. These findings indicate that the quenching process indicate may be caused by quenching process may be caused by dynamic quenching. dynamic quenching.
Figure 7. 7. Stern–Volmer metronidazole under under Figure Stern–Volmer plots plots for for the the system system of of FCNs FCNs in in the the presence presence of of metronidazole 323 K, K, respectively. F0 and Fare Fare the fluorescence intensity of FCNs temperatures of of 293, 293,303, 303,313, 313,and and 323 respectively. F0 and the fluorescence intensity of in the in absence and presence of metronidazole, respectively. Conditions: FCNs, 500 μL;500 PBS, 0.2PBS, M, FCNs the absence and presence of metronidazole, respectively. Conditions: FCNs, µL; pH M, = 7.0. 0.2 pH = 7.0. Table 1. 1. Stern–Volmer interaction of C-Dots and and metronidazole metronidazole at at Stern–Volmer quenching quenching constants constants for for the the interaction of C-Dots Table different temperatures. temperatures. different pH 7.0 7.0 7.0 7.0
pH T(K) 7.0 293 7.0 303 7.0 313 7.0 323
T(K) KSV(L· mol−1) Kq(L· mol−1· S−1) KSV (L·mol−1 ) Kq (L·mol−1 ·S−1 ) 293 3.98 × 103 3.98 × 1011 3 11 3.98 × 10 3.98 3 303 4.26 ×3 10 4.26××10 101111 4.26 × 10 4.26 × 10 11 313 4.684.68 × 103 4.68 × 10 × 103 4.68 × 1011 3 3 323 5.985.98 5.98××10 101111 × 10× 10 5.98
R SD R SD 0.99858 0.01681 0.99858 0.99734 0.02460.01681 0.99734 0.0246 0.99754 0.026010.02601 0.99754 0.99321 0.99321 0.0555 0.0555
3.5. Optimal Conditions for FCNs Detection 3.5. Optimal Conditions for FCNs Detection 3.5.1. Effect of pH pH 3.5.1. Effect of The dependence dependence of of FCNs FCNsfluorescence fluorescenceupon uponpH pHinin the presence MNZ is shown in Figure The the presence of of MNZ is shown in Figure 8A. 8A. In this work, the fluorescence quenching efficiency of MNZ on FCNs is defined as F 0/F, where In this work, the fluorescence quenching efficiency of MNZ on FCNs is defined as F0 /F, where F0 and F F0 and are the fluorescence FCNs in theand presence absence of MNZ, respectively. are the F fluorescence intensitiesintensities of FCNs inofthe presence absenceand of MNZ, respectively. An increase AnpH increase from 7 leadsintothe anfluorescence increase in the fluorescence quenching efficiency of the in from 5 in to pH 7 leads to 5antoincrease quenching efficiency of the system, whereas a system, whereas a further increase in pH results in the decrease in F 0/F. At the same time, the effects further increase in pH results in the decrease in F0 /F. At the same time, the effects of different buffer of different buffer systems on fluorescence quenching efficiency compared. can be systems on fluorescence quenching efficiency were compared. Aswere can be seen fromAs Figure 8B,seen the from Figure 8B, the highestwas quenching was obtained a system. sodium Therefore, phosphatesodium buffer highest quenching efficiency obtainedefficiency from a sodium phosphatefrom buffer system. Therefore, sodium phosphate solution at pH = 7experiments. was selected for the following phosphate buffer solution at pH = 7 was buffer selected for the following experiments.
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Figure Effectof of(A) (A) pH; pH; (B) the potassium phosphate, 2. sodium phosphate and Figure 8. 8.Effect the type typeofofbuffer buffer(1.(1. potassium phosphate, 2. sodium phosphate (C) the dosage ofof FCNs; (D) reaction temperature and (E)(E) reaction time onon thethe and3.3.citrate citratesolution); solution); (C) the dosage FCNs; (D) reaction temperature and reaction time fluorescence intensity theFCNs-MNZ FCNs-MNZsystem. system. Conditions: Conditions: PBS, fluorescence intensity ofof the PBS,0.2 0.2M; M;MNZ, MNZ,125.00 125.00μM. µM.
3.5.2. Effect the DosageofofFCNs FCNs 3.5.2. Effect of of the Dosage The effect of the FCN dosage on the quantitative detection of MNZ is presented in Figure 8C. The effect of the FCN dosage on the quantitative detection of MNZ is presented in Figure 8C. The fluorescence quenching efficiency of the system increased gradually with an increase in the FCN The fluorescence quenching efficiency of the system increased gradually with an increase in the FCN dosage from 300 to 500 μL. When the dosage of FCNs was greater than 500 μL, the quenching dosage from 300 to 500 µL. When the dosage of FCNs was greater than 500 µL, the quenching efficiency efficiency decreased. Consequently, a dosage of 500 μL was used for subsequent experiments. decreased. Consequently, a dosage of 500 µL was used for subsequent experiments. 3.5.3. Effect of Reaction Temperature 3.5.3. Effect of Reaction Temperature The effects of the reaction temperature on the fluorescence quenching efficiency of the system The effects of the reaction temperature on the fluorescence quenching efficiency of the system were studied at 20, 25, 30, 35, and 40 °C. As shown in Figure 8D, the quenching efficiency increased were studied at 20,in25, 30, 35,temperature and 40 ◦ C. As shown 8D, the quenching with an increase reaction from 20 to in 30 Figure °C but decreased thereafter.efficiency Hence, 30increased °C was ◦ C but decreased thereafter. Hence, 30 ◦ C was with an increase in reaction temperature from 20 to 30 selected as the optimum reaction temperature. selected as the optimum reaction temperature. 3.5.4. Effect of Reaction Time 3.5.4. Effect of Reaction Time The effect of reaction time on the fluorescence quenching efficiency of the system is shown in The effect offluorescence reaction time on the fluorescence quenching efficiencyincreased of the system Figure 8E. The quenching efficiency of the system gradually from 1istoshown 10 min in Figure 8E. The fluorescence quenching efficiency of the system gradually increased from 1 to 10 min
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then no significant changes in F0 /F were observed after a reaction time of 10 min. Thus, 10 min was then no significant changes in F0/F were observed after a reaction time of 10 min. Thus, 10 min was considered as the optimum reaction time. considered as the optimum reaction time. 3.6. Selectivity of the Proposed Method 3.6. Selectivity of the Proposed Method To verify the selectivity of the method, we investigated possible interferences from common To verify the selectivity of the method, we investigated possible interferences from common inactive ingredients presented in the drug. As shown in Figure 9, when the concentrations of K++ , Na++ , inactive + ingredients − 2presented − 2− in the drug. As shown in Figure 9, when the concentrations of K , Na , Mg2+ , NH + 2− , CO2− 3 , − SO 3 , glucose, lactose, starch, oxalic acid, glycine and L -histidine were 4 , NO 4 Mg2+, NH , NO , SO , CO 4 3 4 3 , glucose, lactose, starch, oxalic acid, glycine and L-histidine were 200 200 times that of MNZ, no obvious interference was observed. A similar observation was made for times that of MNZ, no obvious interference was observed. A similar observation was made for ascorbic acid and wherewhere their concentrations were 50were times50 that of MNZ. ronidazole ascorbic acid β-cyclodextrin and β-cyclodextrin their concentrations times that For of MNZ. For and secnidazole, however, no interference was observed when the concentrations of the two substances ronidazole and secnidazole, however, no interference was observed when the concentrations of the weretwo comparable with the concentration MNZ. Considering that the quenching of the fluorescence substances were comparable withof the concentration of MNZ. Considering that quenchingcan of also fluorescence be caused bycan other absorption spectra overlapspectra with the emission of alsomolecules be causedwith by other molecules withthat absorption that overlapspectra with the FCNs, we compared absorption spectra between interference, and FCNs (Figures S4–S6). emission spectra UV of FCNs, we compared UV absorption spectraanalytes, between interference, analytes, and The FCNs results(Figures demonstrate quenching due to spectral overlapdue does occur under S4–S6).that Thefluorescence results demonstrate that fluorescence quenching to not spectral overlap selected conditions. the concentrations of ronidazole and secnidazole reachedand five doesexperimental not occur under selected When experimental conditions. When the concentrations of ronidazole reached five times of MNZ, the compromised. All in all, that common timessecnidazole that of MNZ, the results werethat compromised. Allresults in all,were common inactive ingredients exist inactive thatdetection exist in drugs did not affect the detection of MNZ, which indicates in drugs didingredients not affect the of MNZ, which indicates that the method proposed herethat canthe be method proposed here can be used for the selective detection of metronidazole in drugs. Of course, used for the selective detection of metronidazole in drugs. Of course, extra pretreatment steps maybe extrato pretreatment steps maybe requiredintobiological eliminate samples. similar interferences in biological samples. required eliminate similar interferences
+ +, 25 +, Figure 9. Effects of potentially interferingsubstances: substances: (0) (0) non-interference; non-interference; (1) mM; (2)(2) NaNa Figure 9. Effects of potentially interfering (1)KK, +25 , 25 mM; + − 2− 2− 2+ + mM; (5) NO 3 , 25 − 2− (7) CO 3 , 25 mM; 2(8) − glucose, mM; (3) Mg , 25 mM; (4) NH , 25 mM; (6) SO , 25 mM; 2+ 4 4 25 mM; (3) Mg , 25 mM; (4) NH4 , 25 mM; (5) NO3 , 25 mM; (6) SO4 , 25 mM; (7) CO3 , 25 mM; 25 mM;25(9)mM; lactose, 25 mM;25(10) starch, 25 mM; acid, 25 mM; 25 mM; (13) (8) glucose, (9) lactose, mM; (10) starch, 25(11) mM;oxalic (11) oxalic acid, 25 (12) mM;glycine, (12) glycine, 25 mM; L-histidine, 25 mM; (14) ascorbic acid, 6.25 mM; (15) β-cyclodextrine, 6.25 mM; (16) ronidazole, 625 (13) L-histidine, 25 mM; (14) ascorbic acid, 6.25 mM; (15) β-cyclodextrine, 6.25 mM; (16) ronidazole, μM; (17) (17) secnidazole, secnidazole, 625 μL; PBS, 0.20.2 M,M, pHpH 7.0;7.0; MNZ, 125.00 μΜ.µM. 625 µM; 625μM. µM.Conditions: Conditions:FCNs, FCNs,500 500 µL; PBS, MNZ, 125.00
3.7. Fluorescence Detection of MNZ 3.7. Fluorescence Detection of MNZ Figure 10 shows the change in fluorescence intensity of FCNs upon the addition of various Figure 10 shows the change in fluorescence intensity quenching of FCNs upon the addition various concentrations of MNZ. As displayed, the fluorescence efficiency of FCNs of gradually concentrations of MNZ. As displayed, the fluorescence quenching efficiency of FCNs gradually decreased with an increase in the concentration of MNZ. As shown in the upper right inset of decreased an decrease increase in concentration of MNZ. As shown in the aupper inset to of the Figure 10, Figurewith 10, the in the fluorescence quenching efficiency exhibited linearright response MNZ the decrease in fluorescence quenching efficiency exhibited a linear response to the MNZ concentration concentration in the range of 0.8–225.0 μM. The calibration curve can be depicted as F0/F = in the range of 0.8–225.0 µM.concentration The calibration curveμM) can be depicted as F0 /F = 0.0076C+0.9520 (C is the 0.0076C+0.9520 (C is the of MNZ, with a correlation coefficient of 0.9971 and with concentration of MNZ, µM) with a correlation of 0.9971 and with a limit of detection a limit of detection (LOD) at 279 nM. LOD coefficient is defined by the equation LOD = 3S0/K, where S0 (LOD) is the
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at 279 nM.deviation LOD is defined by measurements the equation LOD 3Sand where is theofstandard deviation of blank 0 /K,K standard of blank (n ==7) is theS0slope the calibration curve. The measurements (n = 7) and K is the slope of the calibration curve. The repeatability of the proposed repeatability of the proposed method was also evaluated by performing a series of ten for 125.0 μM method was also evaluated bydeviation performing a series of tenwas for 125.0 µM MNZ and a suggests relative standard MNZ and a relative standard (RSD) of 0.72% obtained. This result that our deviation (RSD) of 0.72% was obtained. This result suggests that our assay protocol is very precise. assay protocol is very precise.
Figure 10. 10. The The change change in influorescence fluorescenceintensity intensityofofFCNs FCNsupon uponthe the addition various concentrations Figure addition of of various concentrations of of MNZ. concentrations of MNZ arefollows: as follows: (a) 0.00; (b) 2.50; (c) (d) 7.50; (d) 15.00; (e) 30.00; (f) MNZ. TheThe concentrations of MNZ are as (a) 0.00; (b) 2.50; (c) 7.50; 15.00; (e) 30.00; (f) 50.00; 50.00; (g) (h) 75.00; (h) 100.00; (i) 125.00; (j) 150.00; (k) 175.00; (l) 200.00; and225.00 (m) 225.00 μM. Conditions: (g) 75.00; 100.00; (i) 125.00; (j) 150.00; (k) 175.00; (l) 200.00; and (m) µM. Conditions: PBS, PBS, M,= pH 7.0; FCNs: 500 μL. 0.2 M,0.2 pH 7.0;=FCNs: 500 µL.
3.8. Applications Applications 3.8. To test test the the feasibility feasibility of of our our protocol, protocol, the the proposed proposed method method was was used used to to determine determine the the To concentration of of MNZ MNZ in in two two different different brands brands of of metronidazole metronidazole tablets tablets and and to to test test the the recovery recovery from from concentration the spiked tablets. The results are depicted in Table 2. The amount of MNZ measured in the tablets the spiked tablets. The results are depicted in Table 2. The amount of MNZ measured in the tablets with our our method method is is comparable comparable to to that that of of the the method method reported reported in in pharmacopoeia pharmacopoeia [35]. [35]. The The recoveries recoveries with of 92.8–104.4% 92.8–104.4%and andRSD RSD 0.44–1.54% were achieved. These results confirm that the proposed of of of 0.44–1.54% were achieved. These results confirm that the proposed sensor sensor provided good precision and accuracy and that it could potentially be used for the detection provided good precision and accuracy and that it could potentially be used for the detection of MNZ of biological MNZ in biological samples. Tablethe 3 presents resultstests of recovery testsplasma. using rabbit plasma. in samples. Table 3 presents results ofthe recovery using rabbit Good recovery Good recovery (95–105%) and(RSD high < precision < 3%)indicating are obtained, indicating the of (95–105%) and high precision 3%) are (RSD obtained, the applicability ofapplicability the developed the developed method in the analysis of biological samples. method in the analysis of biological samples. Table 2. 2. Recovery Recovery and and precision precision of of metronidazole metronidazolein intablets tablets(n (n== 6). 6). Table Detected Spiked 1 Samples Detected Spiked 1 (μM) (μM) Samples (µM) (µM) 15 15 Tablet 1#22.9422.94 ± 0.23 30 30 ± 0.23 Tablet 1# 4545 1515 2#23.9523.95 3030 ± 0.36± 0.36 TabletTablet 2# 4545
Found Found (μM) (µM) 38.39 ± 0.33 38.39 ± 0.33 52.61 ± 0.45 52.61 ± 0.45 65.49 ± 0.96 65.49 ± 0.96 39.60 ± 0.81 39.60 ± 0.81 53.63 ± 1.29 53.63 ± 65.72 ± 1.44 65.72 ± 1.44
Recovery RSD Pharmacopoeia Recovery RSD Pharmacopoeia (%) (%) Method 2 (mg/tablet) (%) (%) Method 2 (mg/tablet) 103.0 0.44 103.0 0.44 98.9 0.50 0.50 22.98 ±22.98 0.26± 0.26 98.9 94.6 1.13 1.13 94.6 104.4 1.02 1.02 104.4 98.9 1.20 1.20 23.87 ±23.87 0.40± 0.40 98.9 92.8 92.8 1.54 1.54
11
Tablet 1#:1#: batch No. 140602, expiry date: 06/2016, Jilin Wantong Pharmaceutical Zhengzhou Wantong Tablet batch No. 140602, expiry date:from 06/2016, from Jilin WantongGroup Pharmaceutical Group pharmaceutical Co., Ltd., Zhengzhou, China; Tablet 2#: batch No. 150201, expiry date: January 2018, from Great Zhengzhou Wantong pharmaceutical Co., Ltd., Zhengzhou, China; Tablet 2#: batch No. 150201, 2 Medicine (China) Co., Ltd., Wuhan, China. UV-vis method.
expiry date: January 2018, from Great Medicine (China) Co., Ltd., Wuhan, China. 2 UV-vis method.
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Table 3. Metronidazole determination in rabbit plasma (n = 6). Concentration (µM) Initial
Added
Found
Recovery (%)
RSD (%)
1
11.24 ± 0.42
10 20 30
21.06 ± 0.58 31.96 ± 0.96 39.98 ± 1.07
98.2 103.6 95.8
2.20 1.86 2.75
2
9.70 ± 0.38
10 20 30
20.17 ± 0.80 29.10 ± 1.28 38.62 ± 1.50
104.7 97.0 96.4
2.41 1.59 2.63
Rabbit Plasma
3.9. Method Comparison A comparison of method performance between this study and others in terms of sensitivity and linear range is presented in Table 4. Our developed assay exhibits a lower LOD compared to most other studies. It is worth mentioning that our method can be an alternative to others for the determination of MNZ in samples as it avoids the use of sophisticated techniques, complicated operations and the requirement of skilled operators. Table 4. Comparison of the proposed method with other methods for the determination of metronidazole. Method
Linear Range (mol L−1 )
Flow-injection chemiluminescence method
10−7 –4.38
Electrochemical detection Electrochemical method Electrochemical technique
1.46 ×
10−3
×
LOD (mol L−1 ) 6.31 ×
10−10
Ref. [36]
1 × 10−9 –2 × 10−6
1 × 10−8
[37]
1 × 10−7 –2.5 × 10−5
4.7 × 10−8
[38]
5 × 10−7 –4 × 10−4
3.7 × 10−7
[39]
10−10 –1.20
9.99 ×
Kinetic spectrophotometric H-point standard addition method
2.92 × 10−5 –1.46 × 10−4
4.85 × 10−6
[41]
Reversed-phase high performance liquid chromatography
5.84 × 10−5 –4.09 × 10−4
1.93 × 10−6
[42]
2.5 × 10−8 –1 × 10−5
6 × 10−9
[43]
7.60 × 10−7 –1.75 × 10−3
7.60 × 10−7
[44]
10−6 –9.93
-
[45]
10−14
[46]
High performance liquid chromatography Spectrophotometric determination Electrochemical sensor Ion Mobility Spectrometry Cathodic stripping voltammetric method Turn-off FCNs
5.84 × 6×
10−14 –4
10−5
×
10−12
×
2.92 × 10−7 –4.09 × 10−4 3.30×
10−10 –4.49
2.5 ×
10−6 –2.25
×
×
2.874 ×
10−10
Electrochemical sensor
Electrochemical reduction
×
10−6
10−6
10−4
2×
[40]
2.57 × 10−8
[47]
2.10 ×
10−10
[10]
2.79 ×
10−7
This work
4. Conclusions Strong blue-emitting fluorescent carbon nanodots (FCNs) using gardenia as a carbon source were synthesized via a simple and green hydrothermal method and their characterizations are elucidated. Without further chemical modification, the synthesized FCNs have been applied to the detection of metronidazole with high sensitivity and selectivity in commercial tablets and rabbit plasma. Our studies have proved that FCNs could be a useful and powerful luminescence tool for chemical analysis. Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8220/18/4/964/s1, Figure S1: Effect of various qualities of gardenia for synthesizing FCNs and the differential dilution ratio on the fluorescence intensity of FCNs solution at 220 ◦ C for 10 h; Figure S2: Fluorescence spectra (A) and fluorescence intensity (B) of C-dots prepared under various reaction times; Figure S3: Fluorescence spectra (A) and fluorescence
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intensity (B) of C-dots prepared under various temperature; Figure S4: Overlapping between Flurescence spetra of FCNs and the UV-vis absorption spectra of ronidazole; Figure S5. Overlapping between Flurescence spetra of FCNs and the UV-vis absorption spectra of secnidazole; Figure S6. Overlapping between Flurescence spetra of FCNs and the UV-vis absorption spectra of glucose, Na+ and Mg2+ ; Figure S7. Overlapping between flurescence spetra of FCNs and the UV-vis absorption spectra of metronidazole. Acknowledgments: This work was supported by the National Natural Science Foundation of China (21777130, 21277109), the Meritocracy Research Funds of China West Normal University (463132) and the Fundamental Research Funds of China West Normal University (416390). Author Contributions: X.Y. and H.X. conceived and designed the experiments; M.L. and Y.Y. performed the experiments; F.T. helped designing the experiments and performed the data analysis; X.Y., H.X. and X.L. wrote the paper; all the authors reviewed and approved the entire manuscript. Conflicts of Interest: The authors declare no conflict of interest.
References 1.
2.
3. 4.
5.
6. 7.
8.
9.
10.
11.
12.
13.
Mueller, J.; Schildknecht, P.; Mueller, N. Metabolism of nitro drugs metronidazole and nitazoxanide in Giardia lamblia: Characterization of a novel nitroreductase (GlNR2). J. Antimicrob. Chemother. 2013, 68, 1781–1789. [CrossRef] [PubMed] Kumar, L.; Jain, A.; Lal, N.; Sarswat, A.; Jangir, S.; Kumar, L.; Singh, V.; Shah, P.; Jain, S.K.; Maikhuri, J.P.; et al. Potentiating Metronidazole Scaffold against Resistant Trichomonas: Design, Synthesis, Biology and 3D-QSAR Analysis. ACS Med. Chem. Lett. 2012, 3, 83–87. [CrossRef] [PubMed] Lamp, K.C.; Freeman, C.D.; Klutman, N.E.; Lacy, M.K. Pharmacokinetics and pharmacodynamics of the nitroimidazole antimicrobials. Clin. Pharmacokinet. 1999, 36, 353–373. [CrossRef] [PubMed] Li, Y.; Liu, Y.; Yang, Y.; Yu, F.; Liu, J.; Song, H.; Liu, J.; Tang, H.; Ye, B.-C.; Sun, Z. Novel Electrochemical Sensing Platform Based on a Molecularly Imprinted Polymer Decorated 3D Nanoporous Nickel Skeleton for Ultrasensitive and Selective Determination of Metronidazole. ACS Appl. Mater. Interfaces 2015, 7, 15474–15480. [CrossRef] [PubMed] Asma Parveen, S.K.; Nalla, C. Development and validation of a simple and rapid RP-HPLC method for the determination of metronidazole and norfloxacin in combined dosage form. Indian J. Res. Pharm. Biotechnol. 2013, 1, 686–691. Wang, J.H. Determination of three nitroimidazole residues in poultry meat by gas chromatography with nitrogen-phosphorus detection. J. Chromatogr. A 2001, 918, 435–438. [CrossRef] Ali, N.W.; Gamal, M.; Abdelkawy, M. Chromatographic methods for simultaneous determination of diiodohydroxyquinoline and Metronidazole in their binary mixture. Pak. J. Pharm. Sci. 2013, 26, 865–871. [PubMed] Saffaj, T.; Charrouf, M.; Abourriche, A.; Aboud, Y.; Bennamara, A.; Berrada, M. Spectrophotometric determination of metronidazole and secnidazole in pharmaceutical preparations based on the formation of dyes. Dyes Pigment. 2005, 70, 259–262. [CrossRef] Gong, F.-C.; Zhang, X.-B.; Guo, C.-C.; Shen, G.-L.; Yu, R.-Q. Amperometric metronidazole sensor based on the supermolecular recognition by metalloporphyrin incorporated in carbon paste electrode. Sensors 2003, 3, 91–100. [CrossRef] Gholivand, M.B.; Torkashvand, M. A novel high selective and sensitive metronidazole voltammetric sensor based on a molecularly imprinted polymer-carbon paste electrode. Talanta 2011, 84, 905–912. [CrossRef] [PubMed] Roy, E.; Maity, S.K.; Patra, S.; Madhuri, R.; Sharma, P.K. A metronidazole-probe sensor based on imprinted biocompatible nanofilm for rapid and sensitive detection of anaerobic protozoan. RSC Adv. 2014, 4, 32881–32893. [CrossRef] Tan, S.; Jiang, J.; Yan, B.; Shen, G.; Yu, R. Preparation of a novel fluorescence probe based on covalent immobilization by emulsion polymerization and its application to the determination of metronidazole. Anal. Chim. Acta 2006, 560, 191–196. [CrossRef] Pasinszki, T.; Krebsz, M.; Tung, T.T.; Losic, D.; Tung, T.T.; Losic, D. Carbon Nanomaterial Based Biosensors for Non-Invasive Detection of Cancer and Disease Biomarkers for Clinical Diagnosis. Sensors 2017, 17, 1919. [CrossRef] [PubMed]
Sensors 2018, 18, 964
14.
15. 16. 17.
18. 19.
20.
21. 22. 23.
24.
25. 26.
27.
28.
29. 30.
31. 32.
33.
14 of 15
Loo, A.H.; Sofer, Z.; Bousa, D.; Ulbrich, P.; Bonanni, A.; Pumera, M. Carboxylic Carbon Quantum Dots as a Fluorescent Sensing Platform for DNA Detection. ACS Appl. Mater. Interfaces 2016, 8, 1951–1957. [CrossRef] [PubMed] Zhang, R.; Chen, W. Nitrogen-doped carbon quantum dots: Facile synthesis and application as a “turn-off” fluorescent probe for detection of Hg2+ ions. Biosens. Bioelectron. 2014, 55, 83–90. [CrossRef] [PubMed] Fan, R.-J.; Sun, Q.; Zhang, L.; Zhang, Y.; Lu, A.-H. Photoluminescent carbon dots directly derived from polyethylene glycol and their application for cellular imaging. Carbon 2014, 71, 87–93. [CrossRef] Ciotta, E.; Paoloni, S.; Richetta, M.; Pizzoferrato, R.; Prosposito, P.; Tagliatesta, P.; Lorecchio, C.; Venditti, I.; Fratoddi, I.; Casciardi, S. Sensitivity to Heavy-Metal Ions of Unfolded Fullerene Quantum Dots. Sensors 2017, 17, 2614. [CrossRef] [PubMed] Hsu, P.C.; Shih, Z.Y.; Lee, C.H.; Chang, H.T. Synthesis and analytical applications of photoluminescent carbon nanodots. Green Chem. 2012, 14, 917–920. [CrossRef] Lu, W.; Qin, X.; Liu, S.; Chang, G.; Zhang, Y.; Luo, Y.; Asiri, A.M.; Al-Youbi, A.O.; Sun, X. Economical, Green Synthesis of Fluorescent Carbon Nanoparticles and Their Use as Probes for Sensitive and Selective Detection of Mercury(II) Ions. Anal. Chem. 2012, 84, 5351–5357. [CrossRef] [PubMed] Yang, Y.; Cui, J.; Zheng, M.; Hu, C.; Tan, S.; Xiao, Y.; Yang, Q.; Liu, Y. One-step synthesis of amino-functionalized fluorescent carbon nanoparticles by hydrothermal carbonization of chitosan. Chem. Commun. 2012, 48, 380–382. [CrossRef] [PubMed] Zhang, B.; Liu, C.Y.; Liu, Y. A Novel One-Step Approach to Synthesize Fluorescent Carbon Nanoparticles. Eur. J. Inorg. Chem. 2010, 2010, 4411–4414. [CrossRef] Xu, H.; Yang, X.; Li, G.; Zhao, C.; Liao, X. Green synthesis of fluorescent carbon dots for selective detection of tartrazine in food samples. J. Agric. Food Chem. 2015, 63, 6707–6714. [CrossRef] [PubMed] Hatamie, A.; Marahel, F.; Sharifat, A. Green synthesis of graphitic carbon nitride nanosheet (g-C3N4) and using it as a label-free fluorosensor for detection of metronidazole via quenching of the fluorescence. Talanta 2018, 176, 518–525. [CrossRef] [PubMed] Mehta, V.N.; Jha, S.; Basu, H.; Singhal, R.K.; Kailasa, S.K. One-step hydrothermal approach to fabricate carbon dots from apple juice for imaging of mycobacterium and fungal cells. Sens. Actuators B 2015, 213, 434–443. [CrossRef] Xiao, Q.; Lu, S.; Huang, C.; Su, W.; Huang, S. Novel N-Doped Carbon Dots/β-Cyclodextrin Nanocomposites for Enantioselective Recognition of Tryptophan Enantiomers. Sensors 2016, 16, 1874. [CrossRef] [PubMed] Dong, Y.; Pang, H.; Yang, H.B.; Guo, C.; Shao, J.; Chi, Y.; Li, C.M.; Yu, T. Carbon-Based Dots Co-doped with Nitrogen and Sulfur for High Quantum Yield and Excitation-Independent Emission. Angew. Chem. Int. Ed. 2013, 52, 7800–7804. [CrossRef] [PubMed] Wang, X.; Cao, L.; Yang, S.-T.; Lu, F.; Meziani, M.J.; Tian, L.; Sun, K.W.; Bloodgood, M.A.; Sun, Y.-P. Bandgap-Like Strong Fluorescence in Functionalized Carbon Nanoparticles. Angew. Chem. Int. Ed. 2010, 49, 5310–5314. [CrossRef] [PubMed] Qu, K.; Wang, J.; Ren, J.; Qu, X. Carbon Dots Prepared by Hydrothermal Treatment of Dopamine as an Effective Fluorescent Sensing Platform for the Label-Free Detection of Iron(III) Ions and Dopamine. Chem. Eur. J. 2013, 19, 7243–7249. [CrossRef] [PubMed] De, B.; Karak, N. A green and facile approach for the synthesis of water soluble fluorescent carbon dots from banana juice. RSC Adv. 2013, 3, 8286–8290. [CrossRef] Tang, L.; Ji, R.; Cao, X.; Lin, J.; Jiang, H.; Li, X.; Teng, K.S.; Luk, C.M.; Zeng, S.; Hao, J.; et al. Deep Ultraviolet Photoluminescence of Water-Soluble Self-Passivated Graphene Quantum Dots. ACS Nano 2012, 6, 5102–5110. [CrossRef] [PubMed] Shang, J.; Ma, L.; Li, J.; Ai, W.; Yu, T.; Gurzadyan, G.G. The origin of fluorescence from graphene oxide. Sci. Rep. 2012, 2, 792. [CrossRef] [PubMed] Mehta, V.N.; Jha, S.; Kailasa, S.K. One-pot green synthesis of carbon dots by using Saccharum officinarum juice for fluorescent imaging of bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) cells. Mater. Sci. Eng. C 2014, 38, 20–27. [CrossRef] [PubMed] Prasannan, A.; Imae, T. One-Pot Synthesis of Fluorescent Carbon Dots from Orange Waste Peels. Ind. Eng. Chem. Res. 2013, 52, 15673–15678. [CrossRef]
Sensors 2018, 18, 964
34.
35. 36. 37.
38. 39.
40.
41.
42. 43. 44.
45.
46.
47.
15 of 15
Park, J.H.; Kumar, N.; Park, D.H.; Kang, M.H.; Uhm, H.S.; Choi, E.H.; Attri, P.; Yusupov, M.; Neyts, E.C.; Verlackt, C.C.W.; et al. A comparative study for the inactivation of multidrug resistance bacteria using dielectric barrier discharge and nano-second pulsed plasma. Sci. Rep. 2015, 5, 13849. [CrossRef] [PubMed] State Pharmacopoeia Committee. Chinese Pharmacopoeia (Part II); Chemical Industry Press: Beijing, China, 2015. Yan, Z.-Y.; Xiao, A.; Lu, H.; Liu, Z.; Chen, J.-Q. Determination of metronidazole by a flow-injection chemiluminescence method using ZnO-doped carbon quantum dots. Carbon 2014, 77, 1197. [CrossRef] Mollamahale, Y.B.; Ghorbani, M.; Ghalkhani, M.; Vossoughi, M.; Dolati, A. Highly sensitive 3D gold nanotube ensembles: Application to electrochemical determination of metronidazole. Electrochim. Acta 2013, 106, 288–292. [CrossRef] Peng, J.; Hou, C.; Hu, X. Determination of metronidazole in pharmaceutical dosage forms based on reduction at graphene and ionic liquid composite film modified electrode. Sens. Actuators B 2012, 169, 81–87. [CrossRef] Gu, Y.; Yan, X.; Liu, W.; Li, C.; Chen, R.; Tang, L.; Zhang, Z.; Yang, M. Biomimetic sensor based on copper-poly(cysteine) film for the determination of metronidazole. Electrochim. Acta 2015, 152, 108–116. [CrossRef] Yuan, L.; Jiang, L.; Hui, T.; Jie, L.; Xu, B.; Feng, Y.; Li, Y. Fabrication of highly sensitive and selective electrochemical sensor by using optimized molecularly imprinted polymers on multi-walled carbon nanotubes for metronidazole measurement. Sens. Actuators B 2015, 206, 647–652. [CrossRef] Issa, M.M.; Nejem, R.A.M.; Abu Shanab, A.M.; Shaat, N.T. Kinetic spectrophotometric H-point standard addition method for the simultaneous determination of diloxanide furoate and metronidazole in binary mixtures and biological fluids. Spectrochim. Acta Part A 2013, 114, 592–598. [CrossRef] [PubMed] Akay, C.; Ozkan, S.A.; Senturk, Z.; Cevheroglu, S. Simultaneous determination of metronidazole and miconazole in pharmaceutical dosage forms by RP-HPLC. Farmaco 2002, 57, 953–957. [CrossRef] Lu, S.; Wu, K.; Dang, X.; Hu, S. Electrochemical reduction and voltammetric determination of metronidazole at a nanomaterial thin film coated glassy carbon electrode. Talanta 2004, 63, 653–657. [CrossRef] [PubMed] Tavakoli, N.; Varshosaz, J.; Dorkoosh, F.; Zargarzadeh, M.R. Development and validation of a simple HPLC method for simultaneous in vitro determination of amoxicillin and metronidazole at single wavelength. J. Pharm. Biomed. Anal. 2007, 43, 325–329. [CrossRef] [PubMed] Saffaj, N.; Saffaj, T.; Charrouf, M.; Ihssane, B.; Abourriche, A.; Abboud, Y.; Bennamara, A. Spectrophotometric determination of metronidazole and secnidazole in pharmaceutical preparations with alpha naphtol as a chromogenic reagent. Anal. Chem. Indian J. 2007, 4, 78–82. Li, Y.; Liu, Y.; Liu, J.; Liu, J.; Tang, H.; Cao, C.; Zhao, D.; Ding, Y. Molecularly imprinted polymer decorated nanoporous gold for highly selective and sensitive electrochemical sensors. Sci. Rep. 2015, 5, 7699. [CrossRef] [PubMed] Jafari, M.T.; Rezaei, B.; Zaker, B. Ion mobility spectrometry as detector for molecular imprinted polymer separation and metronidazole determination in pharmaceutical and human serum samples. Anal. Chem. 2009, 81, 3585–3591. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
