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One-step synthesis of fluorescent carbon dots for sensitive and selective detection of hyperin
Author’s Accepted Manuscript One-step synthesis of fluorescent carbon dots for sensitive and selective detection of hyperin Lizhen Liu, Zhi Mi, Qin Hu, Caiqing Li, Xiaohua Li, Feng Feng www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(18)30427-2 https://doi.org/10.1016/j.talanta.2018.04.065 TAL18604
To appear in: Talanta Received date: 11 February 2018 Revised date: 17 April 2018 Accepted date: 20 April 2018 Cite this article as: Lizhen Liu, Zhi Mi, Qin Hu, Caiqing Li, Xiaohua Li and Feng Feng, One-step synthesis of fluorescent carbon dots for sensitive and selective detection of hyperin, Talanta, https://doi.org/10.1016/j.talanta.2018.04.065 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
One-step synthesis of fluorescent carbon dots for sensitive and selective detection of hyperin Lizhen Liua, Zhi Mia,*, Qin Hub, Caiqing Lia, Xiaohua Lia, Feng Fenga,* a
Shanxi Datong University, Datong 037009, PR China
b
State University of New York at Buffalo, Natural Science Complex, Buffalo, NY 14260, USA
*
Corresponding author: Tel.: +86-352- 7157968; fax: +86-352- 6100028.
[email protected] [email protected]
Abstract In this article, we presented a new rapid, sensitive and selective method for the determination of hyperin (Hyp) based on the fluorescence quenching of fluorescent carbon dots (CDs). The CDs were prepared by simply mixing an aqueous solution of citric acid with diphosphorus pentoxide. This one-step synthetic route is fast and simple with neither high temperature nor complicated synthesis steps is involved. When Hyp was added to CDs solution, the fluorescence intensity of the CDs significantly decreased. The CDs display high selectivity for Hyp over many potentially interfering substances. Under the optimized conditions, a good linear relationship between the fluorescence intensity ratio Fo/F and the concentration of Hyp is obtained in a range of 0.22-55 µM with a detection limit (S/N = 3) of 78.3 nM. The method was successfully applied for the determination of Hyp in fufangmuji granules and human serum samples with recoveries in a range of 93.3-107.0%. This paper highlights the usefulness of CDs as an effective fluorescence probe for the Hyp detection due to its easy preparation, low-cost, excellent photostability, favorable biocompatibility and low cytotoxicity.
1
Graphical Abstract:
Keywords: Carbon dots; Fluorescence detection and hyperin
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1. Introduction Hyperin (Hyp) shown in Fig. S1 is a flavonol glycoside compound which is abundant in clusiaceae, rosaceae, ericaceae and campanulaceae. The present research reveals that Hyp has a variety of pharmacological activities including anti-depressant [1], antioxidants [2,3], anti-inflammatory [4,5] and liver protection [6]. The clinical results show that it has remarkable curative effect for treating cough, canker sore, hypertension and coronary heart [7-11]. Driven by its pharmacological activities and clinical application, it is essential to develop a convenient and sensitive method to determine Hyp. To date, the analytical methods developed for the Hyp detection include capillary electrophoresis (CE) [12-14], high performance liquid chromatography (HPLC) [15-17], chemiluminescence (CL) [18], electrophoretic deposition (EPD) [19] and mass spectrometry (MS) [20]. These analytical methods could offer good precision and accuracy, however they suffer from drawbacks such as expensive instrument, professional and complicated operation, materials and time consumption. Fortunately, the fluorescence method appears to be the most widespread, convenient and promising detection approach compared with other analytical techniques owing to its simple operation, inexpensive cost, high sensitivity, rapid response and real-time monitoring capability. Carbon dots (CDs) are reported to be fluorescent nanomaterials with sizes of less than 10 nm, The CDs were first discovered in 2004 when Xu et al. were purifying single-walled carbon nanotubes [21]. Afterwards, CDs have attracted widespread attention in the academic and industrial fields owing to their outstanding advantages such as chemical inertness, tunable emission, excellent photostability, low toxicity, good biocompatibility and environmental friendly [22-24]. Until now, tremendous efforts have been dedicated to develop efficient synthetic methods to produce CDs with high performance photoluminescence (PL) properties. The preparation 3
methods of CDs are generally classified into two groups: top-down and bottom-up approaches. The top-down approaches contain laser ablation [25], plasma treatment [26,27] and electrochemical oxidation [28,29]. The bottom-up routes consist of solvothermal synthesis [30,31], microwave treatment [32-34], thermal pyrolysis [35-38], hydrothermal carbonization [39-42] and self-catalysis [43]. Although the typical PL characteristics of CDs play a dominant role in leading to a number of their applications, the mechanisms of PL are still under study. Many research groups have studied the PL properties of CDs and proposed various mechanisms such as the recombination of electron-hole pairs, fluorophores with different degrees of π-conjugation, surface states and quantum effect [44-52]. CDs with excellent properties have been extensively applied in many fields, including storage devices [53], catalysis [54,55], optoelectronic devices [56], drug delivery [57] and bioimaging [58]. In addition, many scientific papers have reported the applications of CDs as fluorescence probes to detect various substances and the research results were inspiring. Loo et al. prepared Carboxylic CDs as a fluorescent probe for DNA detection [59]. Liao et al. demonstrated that the N-doped CDs can be used as a novel fluorescent probe for sensing of Au3+ ions [60]. Miao et al. used CDs derived from tomato juice as a fluorescent probe for quantification of carcinoembryonic antigen [61]. Huang et al. applied bis(3-pyridylmethyl)amine-functionalized CDs as a fluorescent probe for detecting glutathione in live cells [62]. Gu et al. used N-doped CDs derived from lotus root as a fluorescent probe for selective and sensitive sensing of Hg2+ [63] and Zou et al. used N, S co-doped CDs from ʟ-cysteine as a new fluorescent probe for the detection of fluazinam in related samples [64]. However, as far as we are aware, CDs have never been used as a fluorescent probe for Hyp detection so far. Herein, we for the first time applied CDs as a fluorescent probe for Hyp detection. The CDs 4
were synthesized by adding an aqueous solution of citric acid to diphosphorus pentoxide. This synthesis process is green, simple and effective. It was found that these CDs can server as an effective fluorescence probe for the selective and sensitive determination of Hyp. The determination of Hyp was based on fluorescence quenching of CDs. Finally, the proposed method was successfully exploited for the determination of Hyp in fufangmuji granules and human serum samples with satisfactory results are achieved. Moreover, the proposed method exhibits several advantages such as ease of operation, low cost, short analysis time and high sensitivity.
2. Experimental 2.1 Materials Hyperin (Hyp), daidzein (Dai), rutin (Rut), chrysophanol (Chr), ferulic acid (Fer), aloeemodin (Alo), rhein (Rhe), puerarin (Pue) and chlorogenic acid (Chl) were bought from Sinopharm Chemical Reagent Co. Ltd, China. Diphosphorus pentoxide (P2O5) was obtained from Tianjin Fuchen Chemical Reagent Company, China. Hydrochloric acid (HCl), glacial acetic acid, sodium hydroxide (NaOH), methanol, acetonitrile, potassium bromide (KBr), sodium dihydrogen phosphate (NaH2PO4·2H2O) and dibasic sodium phosphates (Na2HPO4·7H2O) were bought from Tianjin Chemical Reagent Company, China. Cysteine (Cys), glutamic (Glu), glycine (Gly), threonine (Thr), tyrosine (Tyr), glucose (Glu), glutathione (Glut), alanine (Ala), histidine (His) and ascorbic acid (Asc) were obtained from Aladdin Reagent Co. Ltd, China. CaCl2, Zn(NO3)2, AlCl3, MgCl2, KCl, FeCl3, NaCl, CuSO4, PbCl2, AgNO3, BaCl2 and CrCl3 were purchased from Beijing Chemical Reagent Company, China. Acetonitrile (ACN) and methanol (MeOH) were obtained from Tianjin Kermel Chemical Reagent Company, China. Fufangmuji granules were obtained from Dandong Pharmaceutical Company Limited, China. All of the chemicals have 5
analytical reagent grade or above and can be utilized directly as received without any further processing. Distilled deionized (DDI) water (≥18 MΩ·cm-1) which was obtained from a Millipore Milli-Q direct water purification system (Bedford, MA, USA) was used to prepare all aqueous solutions.
2.2 CDs preparation The CDs samples were prepared by an easy self-catalysis method. The synthesis process is self-controlled and self-promoted without any external treatment. Briefly, 0.5g citric acid was mixed with 1.0 mL DDI water under ultrasound for 20 min and the obtained homogeneous solution was transferred into a 25 mL beaker containing 2.0 g P2O5 without stirring. The reaction mixture rapidly generated heat to drive the reaction and gradually cooled down to room temperature. Then the dark brown crude product was dissolved in DDI water and centrifuged for 15 min at 6000 rpm to remove the undissolved substances. Then, the supernatant was collected and dialyzed against DDI water for 7 days in a dialysis tubing with MWCO of 1000 Da (Spectrum Laboratories, Rancho Dominguez, CA, USA) while stirring and refilling with fresh DDI water every 24 h. Finally, the dried CDs product was obtained after freeze-drying of CDs solution collected from the dialysis tubing.
2.3 Characterization methods The UV-vis absorption spectra were acquired on a spectrometer (Lambda 35, Perkin Elmer, America) at 200-700 nm. The photoluminescence (PL) spectra was performed by using a HITACHI F-2500 spectrofluorophotometer (F-2500, Hitachi, Japan) equipped with a xenon discharge lamp and 1.0 cm quartz cells. The Fourier transform infrared spectroscopy (FTIR) was 6
conducted on a FTIR spectrometer (Nicolet Magna 550, Thermo Scientific, USA). The X-ray photoelectron spectroscopy (XPS) was performed on a X-ray photoelectron spectrometer (Leybold Heraeus SKL-12, China). The TEM images were taken from a field-emission transmission electron microscope (FEI Tecnai F-20, Hillsboro, USA) operating at 200 kV.
2.4 Preparation of the standard and real samples The standard stock solution of Hyp (2.2 mM) was prepared by dissolving 10 mg Hyp in 10 mL MeOH and stored in the dark at 4 ℃ before use. The phosphate buffered saline (PBS) solutions were prepared by mixing solutions of 0.01 M NaH2PO4 and 0.01 M Na2HPO4 and adjusting the different pH values with 1 M NaOH or 1 M HCl. For fufangmuji granules sample, 1.0 g of the powder was accurately weighed. The weighed sample was extracted with 10 mL of MeOH for 1 h in an ultrasonic bath. The extracted solution was subjected to centrifugation at 8,000 rpm for 5 min. After centrifugation, the supernatant was transferred into a volumetric flask for further analysis. Human blood samples were obtained from Affiliated Hospital of Shanxi Datong University. To isolate the serum, the human blood samples were centrifuged for 15 min at 6000 rpm. A 1.00 mL aliquot of serum sample was mixed with 1.00 mL of ACN in order to remove the proteins and then centrifuged for 30 min at 8000 rpm. After centrifugation, the supernatant solution was collected and the residue of ACN was evaporated by nitrogen flow and stored at 4 oC for further analysis.
2.5 Fluorescence assay of Hyp For the detection of Hyp, 0.2 mL of CDs solution (1.5 mg/mL) was added to 1.8 mL of PBS 7
and then different amounts of Hyp or sample solution were added to the above mixed solutions. The excitation and emission slit widths were set at 10 and 5 nm, respectively. The PL emission spectra (300-540 nm) were recorded after 10 min at 280 nm excitation wavelength (λex). The same conditions were performed for all of the fluorescence detections. The signal output results were calculated according to the relative fluorescence intensities (F0/F), in which F0 and F represent the fluorescence intensity of the CDs in the absence and presence of Hyp, respectively. The selectivity and sensitivity measurements were carried out in triplicate.
3. Results and discussion 3.1 Characterization of CDs TEM was used to study the morphology and size of the as-synthesized CDs. Fig. 1 A and B show the morphology and the size distribution of the as-synthesized CDs. The as-synthesized CDs have nearly spherical shapes and are evenly distributed without obvious aggregation. The results show that the as-synthesized CDs display a size range of 5.0-10.2 nm with a mean diameter of 7.8 nm by randomly counting 100 random particles. The UV-vis absorption and PL spectroscopy were used to study the spectral properties of the as-synthesized CDs. As displayed in Fig. S2, the as-synthesized CDs had two absorption peaks at around 210 nm and 300 nm. The absorption peak at around 210 nm is originated from the π → π* transition of the C=C [65] and the absorption peak at around 300 nm is originated from the n → π* transitions of C=O [66,67]. The CDs shows a strong emission peak at 390 nm and a weak emission peak at 310 nm with a λex of 280 nm. As indicated by the inset of Fig. S2, the CDs solution exhibit strong blue emission under UV light. The emission quantum yield (ΦS) of the as-synthesized CDs is 5.78 % using tryptophan as a reference (Fig. S3). The PL properties of the 8
as-synthesized CDs were studied under different λex. As displayed in Fig. S4, the emission wavelength (λem) is red shifted when the λex changes from 280 nm to 500 nm. It indicates that the as-prepared CDs have λex-dependent emission behavior, which has commonly observed in the other previous reported CDs [68-72]. This behavior may be due to the different distribution of emissive energy trap, different size distribution of CDs and different surface states on CDs [73-74]. IR spectra was obtained to investigate the surface functionality of the as-prepared CDs and depicted in Fig. S5. The broad absorption band at around 3413 cm-1 corresponds to the O‒H stretching vibration. The peaks at 2937 and 2850 cm-1 are attributed to the C‒H stretching vibration. The characteristic peaks at 1720, 1639 and 1402 cm-1 correspond to the C=O, C=C and O=C-O stretching vibration, respectively [75]. The other peaks at 2362 cm-1 correspond to P-OH bending vibration of phosphate, indicating presence of phosphate moiety on the surface of CDs. Several peaks at 1093, 993 and 914 cm-1 reveal the existence of P=O, P-O-C and P-O-H groups, respectively [76]. In addition, more information on the surface chemical and element compositions of as-prepared CDs is further revealed by XPS analysis. As displayed in Fig. S6A, the XPS spectra show that the as-prepared CDs are mainly comprised of carbon, oxygen and phosphorus. The binding energy peaks at 284.8, 532.4 and 135.1 eV were attributed to C1s, O1s, and P2p, respectively. Specifically, the C1s spectra (Fig. S6B) splits into three peaks at 284.8, 286.6 and 288.3 eV, which can be assigned to C=C, C-OH and C=O functional groups, respectively [77-79]. The O1s spectra (Fig. S6C) demonstrates two peaks at 532.0 and 533.2 eV, which can be related to C=O/O=C-OH and C-O-C/C-OH functional groups, respectively [78,80,81]. The P2p spectra (Fig. S6D) displays one peak at 135.1 eV for PO43-. In summary, the IR and XPS results indicate that the surface of the as-synthesized CDs is functionalized with 9
carbonyl group, carboxyl group, hydroxyl group and phosphate moiety.
3.2 Optimization for Hyp detection In order to make the as-prepared CDs an effective fluorescence probe for Hyp detection, the critical parameters including the concentration of CDs, pH and reaction time between CDs and Hyp were optimized. The first attempt was given to study the influence of CDs concentration (0.04-0.23 mg/mL) on the relative fluorescence intensity (Fo/F), where F and Fo are the fluorescent intensities of CDs with and without Hyp, respectively. As shown in Fig. S7, the Fo/F increases when increasing the concentration of the CDs to 0.15 mg/mL and then decreases when the concentration of the CDs is over 0.15 mg/mL. Thus, the 0.15 mg/mL was selected as the optimum CDs concentration for the detection of Hyp. The influence of pH on the fluorescent intensity of sensor system was researched in the pH range of 2.0-12.0. Fig. 2A(1) shows the fluorescence intensity of CDs under different pH without Hyp. The fluorescence intensity of CDs increases steadily to maximum when the pH is increased from 2.0 to 8.0 and later decreases when further increasing the pH value. This indicates that pH-dependent fluorescence intensity of CDs. Fig. 2A(2) exhibits the fluorescence intensity of CDs under different pH with 5.5 µM Hyp. The result shows no obvious change in the fluorescence intensity, rather a slight increment with the increase of pH up to pH 8.0 and then a slight decrement with the pH changed from 8.0 to 12.0. The Fo/F at different pH is illustrated in Fig. 2B. It can be seen that the Fo/F is the largest at pH 8.0 and therefore 8.0 was selected as the optimal pH value for the subsequent study. The effect of reaction time between CDs and Hyp was studied and depicted in Fig. S8. The 10
fluorescence intensity decreases dramatically within one minute, then slightly decreases and finally remains nearly constant after 10 min. As such, 10 min was selected for the following experiment.
3.3 Study of potential interferences To examine the capability of our proposed method for real samples analysis, the effects of potential interfering substances, including relevant metal ions (Al3+, Zn2+, Fe3+, Na+, Ca2+, Mg2+, K+, Cu2+, Pb2+, Ag+, Ba2+ and Cr3+), biomolecules (cysteine, glutamic acid, glycine, threonine, tyrosine, glucose, glutathione, alanine, histidine and ascorbic acid) and other co-existing compounds (chrysophanol, rutin, daidzein, ferulic acid, aloeemodin, rhein, puerarin and chlorogenic acid) which may be present in real samples, were examined. As shown in Fig. 3, Hyp causes obvious change in fluorescence intensity of CDs while the other interfering substances have low or negligible influence on the fluorescence of CDs, indicating that the CDs show an excellent selectivity toward Hyp.
3.4 Analytical performance Under the above optimized conditions, the fluorescent quenching of CDs by different concentration of Hyp was investigated. As shown by Fig. 4A, the fluorescence intensity of CDs decreases gradually with the increase of Hyp concentration. This reveals that the fluorescence intensity of CDs is highly correlated with the Hyp concentration. The fluorescence quenching of CDs by Hyp can be expressed by the Stern-Volmer equation [82,83]: Fo/F = KSV[C] + 1
(1)
where KSV is the Stern-Volmer constant, [C] is the concentration of Hyp, F and Fo represent the 11
fluorescence intensities of CDs with and without Hyp, respectively. A Stern-Volmer plot is displayed in Fig. 4B and the linear regression equation of Fo/F = 0.0361[C] + 1 is obtained. An excellent linear relationship between Fo/F and Hyp concentration is attained over the concentration range of 0.22-55 µM and the correlation coefficient (r) is 0.9983. The limit of detection (LOD) is calculated to be 78.3 nM (S/N = 3). These results indicate that the proposed CDs-based nanosensor
is very useful for the detection of Hyp.
A comparison of our proposed method with some other previous reported methods for Hyp detection was studied and the results are summarized in Table S1. The LOD obtained with our developed method was lower than that reported methods [15,17,19,84-86] except the chemiluminescence method [18]. However, our proposed CDs-based nanosensor is simple, rapid and cost-effective. Moreover, the precision and accuracy of this developed method were evaluated. Ten repeated measurements of 11, 22 and 44 µM Hyp under the same conditions were performed and the results were depicted in Table S2. The recoveries and the RSD are 97.3-101.8% and 1.2-1.7%, respectively. The results indicate that our developed method have good accuracy and precision.
3.5 Possible sensing mechanism As indicated by Fig. 4A, the fluorescence intensity of the CDs decreases significantly with the increase of Hyp concentrations. Meanwhile, a red shift from 390 to 440 nm of the emission spectrum was observed. Since the surface of CDs are covered with abundant basic groups such as carboxyl and hydroxyl groups as determined by FTIR (Fig. S5) and XPS (Fig. S6), it is highly possible that the phenolic hydroxyl groups of Hyp bind to the carboxyl and hydroxyl groups of CDs through hydrogen bonding, resulting in the change in the surface of CDs. It is known that 12
CDs are good electron donors and acceptors [87]. As such, there is a possible to promote an electron transfer from the photo-excited CDs to the aromatic groups of Hyp. In another word, the CDs are wrapped by the Hyp mimicking dendrimers, allowing an effective non-radiative energy transfer from the CDs to Hyp, resulting in quenching of CDs [88].
3.6 Sample analysis The proposed method was finally used for the detection of Hyp in real samples, i.e. fufangmuji granules and human serum samples, by using the standard addition method. Table 1 summarizes the analytical results of Hyp in fufangmuji granules and human serum samples. The recoveries of Hyp in the fufangmuji granules and human serum samples are in the range of 93.3-107 % with the RSD (n = 5) are in the range of 1.16-1.72 %. The results reveal that our proposed method can be applied to detect Hyp in real samples with high precision and good repeatability.
4.0 Conclusion In this work, we developed a novel, simple, selective and sensitive method for the determination of Hyp based on the fluorescence quenching of CDs. The preparation of CDs is simple, green and economic. Compared with the reported methods for Hyp determination, our proposed method shows some advantages. First, this method is simple, economic and efficient without expensive instrument and complex operation. Second, this CDs-based fluorescence sensor has excellent sensitivity and good selectivity. Third, there is no need for further chemical modification of CDs, and all the chemicals and materials employed in this work are nontoxic or of low toxicity, making the process environmental-friendly. Furthermore, the developed method was used to detect Hyp in fufangmuji granules and human serum samples with satisfactory results 13
achieved. Our work not only adds to the database of the Hyp determination methods, but also reflects the potential application of CDs as nanosensors.
Acknowledgments The authors gratefully acknowledge the support from the National Nature Science Foundation of China (21375083).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. talanta.
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21
(A)
(B)
Fig. 1. (A) TEM image of the as-prepared CDs. (B) The particle size distribution of the as-prepared CDs.
22
(A)
(B)
Fig. 2. (A) The fluorescence intensity of CDs under different pH (1) in the absence and (2) presence of Hyp. (B) Effect of pH on the relative fluorescence intensity (Fo/F) of CDs. 0.15 mg/mL CDs and 5.5 µM Hyp were used. The error bars represent the standard deviation of the three independent measurements.
23
(A)
(B)
(C)
Fig. 3. Effects of (A) relevant metal ions, (B) biomolecules and (C) other co-existing compounds on Fo/F of CDs. 0.15 mg/mL CDs and 44 µM Hyp were used. The concentration of interfering substances are 50 µM for K+, Na+, Ca2+, Mg2+, Fe3+, Al3+, Zn2+, Cu2+, Pb2+, Ag+, Ba2+, Cr3+, Cys and Glu, 300 µM for Thr, Gly, Try, Glu, Glut, Ala, His and Asc, 100 µM for Rut, Chr, Dai, Fer, Alo, Rhe, Pue and Chl, respectively. The error bars represent the standard deviation of three independent measurements.
24
(A)
(B)
Fig. 4. (A) Fluorescence spectra of CDs in the presence of different concentrations of Hyp, from top to bottom, the concentration of Hyp are 0.00, 0.22, 1.00, 2.20, 5.50, 11.0, 16.5, 22.0, 27.5, 33.0, 38.5, 44.0, 49.5 and 55.0 µM. (B) Stern-Volmer plot of Fo/F versus Hyp concentration. 0.15 mg/mL CDs was used. The error bars represent the standard deviation of the three independent measurements.
25
Table 1 Analytical results of Hyp in real samples Sample
Fufangmuji granules
Human serum
Added (mg/g) 0.00 0.15 0.35 0.55
Found (mg/g) 0.62 0.76 0.98 1.21
Recovery (%, n = 5) ‒ 93.3 103 107
RSD (%, n = 5) 1.42 1.72 1.53 1.66
Added (mM) 0.20 0.40 0.50
Found (mM) 0.21 0.38 0.52
Recovery (%, n = 5) 105 95 104
RSD (%, n = 5) 1.37 1.46 1.28
26
Highlights
A novel sensitive and convenient method for determination of hyperin based on the fluorescence quenching of fluorescent carbon dots (CDs) was developed.
The CDs was used for determination of hyperin with a detection limit of 78.3 nM.
The CDs exhibited excellent sensitivity and selectivity to the detection of hyperin.
The method has satisfactory results for the determination of hyperin in real samples.
This work not only provides a new method for the detection of hyperin but also enriches the application of CDs.
27
PII: DOI: Reference:
S0039-9140(18)30427-2 https://doi.org/10.1016/j.talanta.2018.04.065 TAL18604
To appear in: Talanta Received date: 11 February 2018 Revised date: 17 April 2018 Accepted date: 20 April 2018 Cite this article as: Lizhen Liu, Zhi Mi, Qin Hu, Caiqing Li, Xiaohua Li and Feng Feng, One-step synthesis of fluorescent carbon dots for sensitive and selective detection of hyperin, Talanta, https://doi.org/10.1016/j.talanta.2018.04.065 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
One-step synthesis of fluorescent carbon dots for sensitive and selective detection of hyperin Lizhen Liua, Zhi Mia,*, Qin Hub, Caiqing Lia, Xiaohua Lia, Feng Fenga,* a
Shanxi Datong University, Datong 037009, PR China
b
State University of New York at Buffalo, Natural Science Complex, Buffalo, NY 14260, USA
*
Corresponding author: Tel.: +86-352- 7157968; fax: +86-352- 6100028.
[email protected] [email protected]
Abstract In this article, we presented a new rapid, sensitive and selective method for the determination of hyperin (Hyp) based on the fluorescence quenching of fluorescent carbon dots (CDs). The CDs were prepared by simply mixing an aqueous solution of citric acid with diphosphorus pentoxide. This one-step synthetic route is fast and simple with neither high temperature nor complicated synthesis steps is involved. When Hyp was added to CDs solution, the fluorescence intensity of the CDs significantly decreased. The CDs display high selectivity for Hyp over many potentially interfering substances. Under the optimized conditions, a good linear relationship between the fluorescence intensity ratio Fo/F and the concentration of Hyp is obtained in a range of 0.22-55 µM with a detection limit (S/N = 3) of 78.3 nM. The method was successfully applied for the determination of Hyp in fufangmuji granules and human serum samples with recoveries in a range of 93.3-107.0%. This paper highlights the usefulness of CDs as an effective fluorescence probe for the Hyp detection due to its easy preparation, low-cost, excellent photostability, favorable biocompatibility and low cytotoxicity.
1
Graphical Abstract:
Keywords: Carbon dots; Fluorescence detection and hyperin
.
2
1. Introduction Hyperin (Hyp) shown in Fig. S1 is a flavonol glycoside compound which is abundant in clusiaceae, rosaceae, ericaceae and campanulaceae. The present research reveals that Hyp has a variety of pharmacological activities including anti-depressant [1], antioxidants [2,3], anti-inflammatory [4,5] and liver protection [6]. The clinical results show that it has remarkable curative effect for treating cough, canker sore, hypertension and coronary heart [7-11]. Driven by its pharmacological activities and clinical application, it is essential to develop a convenient and sensitive method to determine Hyp. To date, the analytical methods developed for the Hyp detection include capillary electrophoresis (CE) [12-14], high performance liquid chromatography (HPLC) [15-17], chemiluminescence (CL) [18], electrophoretic deposition (EPD) [19] and mass spectrometry (MS) [20]. These analytical methods could offer good precision and accuracy, however they suffer from drawbacks such as expensive instrument, professional and complicated operation, materials and time consumption. Fortunately, the fluorescence method appears to be the most widespread, convenient and promising detection approach compared with other analytical techniques owing to its simple operation, inexpensive cost, high sensitivity, rapid response and real-time monitoring capability. Carbon dots (CDs) are reported to be fluorescent nanomaterials with sizes of less than 10 nm, The CDs were first discovered in 2004 when Xu et al. were purifying single-walled carbon nanotubes [21]. Afterwards, CDs have attracted widespread attention in the academic and industrial fields owing to their outstanding advantages such as chemical inertness, tunable emission, excellent photostability, low toxicity, good biocompatibility and environmental friendly [22-24]. Until now, tremendous efforts have been dedicated to develop efficient synthetic methods to produce CDs with high performance photoluminescence (PL) properties. The preparation 3
methods of CDs are generally classified into two groups: top-down and bottom-up approaches. The top-down approaches contain laser ablation [25], plasma treatment [26,27] and electrochemical oxidation [28,29]. The bottom-up routes consist of solvothermal synthesis [30,31], microwave treatment [32-34], thermal pyrolysis [35-38], hydrothermal carbonization [39-42] and self-catalysis [43]. Although the typical PL characteristics of CDs play a dominant role in leading to a number of their applications, the mechanisms of PL are still under study. Many research groups have studied the PL properties of CDs and proposed various mechanisms such as the recombination of electron-hole pairs, fluorophores with different degrees of π-conjugation, surface states and quantum effect [44-52]. CDs with excellent properties have been extensively applied in many fields, including storage devices [53], catalysis [54,55], optoelectronic devices [56], drug delivery [57] and bioimaging [58]. In addition, many scientific papers have reported the applications of CDs as fluorescence probes to detect various substances and the research results were inspiring. Loo et al. prepared Carboxylic CDs as a fluorescent probe for DNA detection [59]. Liao et al. demonstrated that the N-doped CDs can be used as a novel fluorescent probe for sensing of Au3+ ions [60]. Miao et al. used CDs derived from tomato juice as a fluorescent probe for quantification of carcinoembryonic antigen [61]. Huang et al. applied bis(3-pyridylmethyl)amine-functionalized CDs as a fluorescent probe for detecting glutathione in live cells [62]. Gu et al. used N-doped CDs derived from lotus root as a fluorescent probe for selective and sensitive sensing of Hg2+ [63] and Zou et al. used N, S co-doped CDs from ʟ-cysteine as a new fluorescent probe for the detection of fluazinam in related samples [64]. However, as far as we are aware, CDs have never been used as a fluorescent probe for Hyp detection so far. Herein, we for the first time applied CDs as a fluorescent probe for Hyp detection. The CDs 4
were synthesized by adding an aqueous solution of citric acid to diphosphorus pentoxide. This synthesis process is green, simple and effective. It was found that these CDs can server as an effective fluorescence probe for the selective and sensitive determination of Hyp. The determination of Hyp was based on fluorescence quenching of CDs. Finally, the proposed method was successfully exploited for the determination of Hyp in fufangmuji granules and human serum samples with satisfactory results are achieved. Moreover, the proposed method exhibits several advantages such as ease of operation, low cost, short analysis time and high sensitivity.
2. Experimental 2.1 Materials Hyperin (Hyp), daidzein (Dai), rutin (Rut), chrysophanol (Chr), ferulic acid (Fer), aloeemodin (Alo), rhein (Rhe), puerarin (Pue) and chlorogenic acid (Chl) were bought from Sinopharm Chemical Reagent Co. Ltd, China. Diphosphorus pentoxide (P2O5) was obtained from Tianjin Fuchen Chemical Reagent Company, China. Hydrochloric acid (HCl), glacial acetic acid, sodium hydroxide (NaOH), methanol, acetonitrile, potassium bromide (KBr), sodium dihydrogen phosphate (NaH2PO4·2H2O) and dibasic sodium phosphates (Na2HPO4·7H2O) were bought from Tianjin Chemical Reagent Company, China. Cysteine (Cys), glutamic (Glu), glycine (Gly), threonine (Thr), tyrosine (Tyr), glucose (Glu), glutathione (Glut), alanine (Ala), histidine (His) and ascorbic acid (Asc) were obtained from Aladdin Reagent Co. Ltd, China. CaCl2, Zn(NO3)2, AlCl3, MgCl2, KCl, FeCl3, NaCl, CuSO4, PbCl2, AgNO3, BaCl2 and CrCl3 were purchased from Beijing Chemical Reagent Company, China. Acetonitrile (ACN) and methanol (MeOH) were obtained from Tianjin Kermel Chemical Reagent Company, China. Fufangmuji granules were obtained from Dandong Pharmaceutical Company Limited, China. All of the chemicals have 5
analytical reagent grade or above and can be utilized directly as received without any further processing. Distilled deionized (DDI) water (≥18 MΩ·cm-1) which was obtained from a Millipore Milli-Q direct water purification system (Bedford, MA, USA) was used to prepare all aqueous solutions.
2.2 CDs preparation The CDs samples were prepared by an easy self-catalysis method. The synthesis process is self-controlled and self-promoted without any external treatment. Briefly, 0.5g citric acid was mixed with 1.0 mL DDI water under ultrasound for 20 min and the obtained homogeneous solution was transferred into a 25 mL beaker containing 2.0 g P2O5 without stirring. The reaction mixture rapidly generated heat to drive the reaction and gradually cooled down to room temperature. Then the dark brown crude product was dissolved in DDI water and centrifuged for 15 min at 6000 rpm to remove the undissolved substances. Then, the supernatant was collected and dialyzed against DDI water for 7 days in a dialysis tubing with MWCO of 1000 Da (Spectrum Laboratories, Rancho Dominguez, CA, USA) while stirring and refilling with fresh DDI water every 24 h. Finally, the dried CDs product was obtained after freeze-drying of CDs solution collected from the dialysis tubing.
2.3 Characterization methods The UV-vis absorption spectra were acquired on a spectrometer (Lambda 35, Perkin Elmer, America) at 200-700 nm. The photoluminescence (PL) spectra was performed by using a HITACHI F-2500 spectrofluorophotometer (F-2500, Hitachi, Japan) equipped with a xenon discharge lamp and 1.0 cm quartz cells. The Fourier transform infrared spectroscopy (FTIR) was 6
conducted on a FTIR spectrometer (Nicolet Magna 550, Thermo Scientific, USA). The X-ray photoelectron spectroscopy (XPS) was performed on a X-ray photoelectron spectrometer (Leybold Heraeus SKL-12, China). The TEM images were taken from a field-emission transmission electron microscope (FEI Tecnai F-20, Hillsboro, USA) operating at 200 kV.
2.4 Preparation of the standard and real samples The standard stock solution of Hyp (2.2 mM) was prepared by dissolving 10 mg Hyp in 10 mL MeOH and stored in the dark at 4 ℃ before use. The phosphate buffered saline (PBS) solutions were prepared by mixing solutions of 0.01 M NaH2PO4 and 0.01 M Na2HPO4 and adjusting the different pH values with 1 M NaOH or 1 M HCl. For fufangmuji granules sample, 1.0 g of the powder was accurately weighed. The weighed sample was extracted with 10 mL of MeOH for 1 h in an ultrasonic bath. The extracted solution was subjected to centrifugation at 8,000 rpm for 5 min. After centrifugation, the supernatant was transferred into a volumetric flask for further analysis. Human blood samples were obtained from Affiliated Hospital of Shanxi Datong University. To isolate the serum, the human blood samples were centrifuged for 15 min at 6000 rpm. A 1.00 mL aliquot of serum sample was mixed with 1.00 mL of ACN in order to remove the proteins and then centrifuged for 30 min at 8000 rpm. After centrifugation, the supernatant solution was collected and the residue of ACN was evaporated by nitrogen flow and stored at 4 oC for further analysis.
2.5 Fluorescence assay of Hyp For the detection of Hyp, 0.2 mL of CDs solution (1.5 mg/mL) was added to 1.8 mL of PBS 7
and then different amounts of Hyp or sample solution were added to the above mixed solutions. The excitation and emission slit widths were set at 10 and 5 nm, respectively. The PL emission spectra (300-540 nm) were recorded after 10 min at 280 nm excitation wavelength (λex). The same conditions were performed for all of the fluorescence detections. The signal output results were calculated according to the relative fluorescence intensities (F0/F), in which F0 and F represent the fluorescence intensity of the CDs in the absence and presence of Hyp, respectively. The selectivity and sensitivity measurements were carried out in triplicate.
3. Results and discussion 3.1 Characterization of CDs TEM was used to study the morphology and size of the as-synthesized CDs. Fig. 1 A and B show the morphology and the size distribution of the as-synthesized CDs. The as-synthesized CDs have nearly spherical shapes and are evenly distributed without obvious aggregation. The results show that the as-synthesized CDs display a size range of 5.0-10.2 nm with a mean diameter of 7.8 nm by randomly counting 100 random particles. The UV-vis absorption and PL spectroscopy were used to study the spectral properties of the as-synthesized CDs. As displayed in Fig. S2, the as-synthesized CDs had two absorption peaks at around 210 nm and 300 nm. The absorption peak at around 210 nm is originated from the π → π* transition of the C=C [65] and the absorption peak at around 300 nm is originated from the n → π* transitions of C=O [66,67]. The CDs shows a strong emission peak at 390 nm and a weak emission peak at 310 nm with a λex of 280 nm. As indicated by the inset of Fig. S2, the CDs solution exhibit strong blue emission under UV light. The emission quantum yield (ΦS) of the as-synthesized CDs is 5.78 % using tryptophan as a reference (Fig. S3). The PL properties of the 8
as-synthesized CDs were studied under different λex. As displayed in Fig. S4, the emission wavelength (λem) is red shifted when the λex changes from 280 nm to 500 nm. It indicates that the as-prepared CDs have λex-dependent emission behavior, which has commonly observed in the other previous reported CDs [68-72]. This behavior may be due to the different distribution of emissive energy trap, different size distribution of CDs and different surface states on CDs [73-74]. IR spectra was obtained to investigate the surface functionality of the as-prepared CDs and depicted in Fig. S5. The broad absorption band at around 3413 cm-1 corresponds to the O‒H stretching vibration. The peaks at 2937 and 2850 cm-1 are attributed to the C‒H stretching vibration. The characteristic peaks at 1720, 1639 and 1402 cm-1 correspond to the C=O, C=C and O=C-O stretching vibration, respectively [75]. The other peaks at 2362 cm-1 correspond to P-OH bending vibration of phosphate, indicating presence of phosphate moiety on the surface of CDs. Several peaks at 1093, 993 and 914 cm-1 reveal the existence of P=O, P-O-C and P-O-H groups, respectively [76]. In addition, more information on the surface chemical and element compositions of as-prepared CDs is further revealed by XPS analysis. As displayed in Fig. S6A, the XPS spectra show that the as-prepared CDs are mainly comprised of carbon, oxygen and phosphorus. The binding energy peaks at 284.8, 532.4 and 135.1 eV were attributed to C1s, O1s, and P2p, respectively. Specifically, the C1s spectra (Fig. S6B) splits into three peaks at 284.8, 286.6 and 288.3 eV, which can be assigned to C=C, C-OH and C=O functional groups, respectively [77-79]. The O1s spectra (Fig. S6C) demonstrates two peaks at 532.0 and 533.2 eV, which can be related to C=O/O=C-OH and C-O-C/C-OH functional groups, respectively [78,80,81]. The P2p spectra (Fig. S6D) displays one peak at 135.1 eV for PO43-. In summary, the IR and XPS results indicate that the surface of the as-synthesized CDs is functionalized with 9
carbonyl group, carboxyl group, hydroxyl group and phosphate moiety.
3.2 Optimization for Hyp detection In order to make the as-prepared CDs an effective fluorescence probe for Hyp detection, the critical parameters including the concentration of CDs, pH and reaction time between CDs and Hyp were optimized. The first attempt was given to study the influence of CDs concentration (0.04-0.23 mg/mL) on the relative fluorescence intensity (Fo/F), where F and Fo are the fluorescent intensities of CDs with and without Hyp, respectively. As shown in Fig. S7, the Fo/F increases when increasing the concentration of the CDs to 0.15 mg/mL and then decreases when the concentration of the CDs is over 0.15 mg/mL. Thus, the 0.15 mg/mL was selected as the optimum CDs concentration for the detection of Hyp. The influence of pH on the fluorescent intensity of sensor system was researched in the pH range of 2.0-12.0. Fig. 2A(1) shows the fluorescence intensity of CDs under different pH without Hyp. The fluorescence intensity of CDs increases steadily to maximum when the pH is increased from 2.0 to 8.0 and later decreases when further increasing the pH value. This indicates that pH-dependent fluorescence intensity of CDs. Fig. 2A(2) exhibits the fluorescence intensity of CDs under different pH with 5.5 µM Hyp. The result shows no obvious change in the fluorescence intensity, rather a slight increment with the increase of pH up to pH 8.0 and then a slight decrement with the pH changed from 8.0 to 12.0. The Fo/F at different pH is illustrated in Fig. 2B. It can be seen that the Fo/F is the largest at pH 8.0 and therefore 8.0 was selected as the optimal pH value for the subsequent study. The effect of reaction time between CDs and Hyp was studied and depicted in Fig. S8. The 10
fluorescence intensity decreases dramatically within one minute, then slightly decreases and finally remains nearly constant after 10 min. As such, 10 min was selected for the following experiment.
3.3 Study of potential interferences To examine the capability of our proposed method for real samples analysis, the effects of potential interfering substances, including relevant metal ions (Al3+, Zn2+, Fe3+, Na+, Ca2+, Mg2+, K+, Cu2+, Pb2+, Ag+, Ba2+ and Cr3+), biomolecules (cysteine, glutamic acid, glycine, threonine, tyrosine, glucose, glutathione, alanine, histidine and ascorbic acid) and other co-existing compounds (chrysophanol, rutin, daidzein, ferulic acid, aloeemodin, rhein, puerarin and chlorogenic acid) which may be present in real samples, were examined. As shown in Fig. 3, Hyp causes obvious change in fluorescence intensity of CDs while the other interfering substances have low or negligible influence on the fluorescence of CDs, indicating that the CDs show an excellent selectivity toward Hyp.
3.4 Analytical performance Under the above optimized conditions, the fluorescent quenching of CDs by different concentration of Hyp was investigated. As shown by Fig. 4A, the fluorescence intensity of CDs decreases gradually with the increase of Hyp concentration. This reveals that the fluorescence intensity of CDs is highly correlated with the Hyp concentration. The fluorescence quenching of CDs by Hyp can be expressed by the Stern-Volmer equation [82,83]: Fo/F = KSV[C] + 1
(1)
where KSV is the Stern-Volmer constant, [C] is the concentration of Hyp, F and Fo represent the 11
fluorescence intensities of CDs with and without Hyp, respectively. A Stern-Volmer plot is displayed in Fig. 4B and the linear regression equation of Fo/F = 0.0361[C] + 1 is obtained. An excellent linear relationship between Fo/F and Hyp concentration is attained over the concentration range of 0.22-55 µM and the correlation coefficient (r) is 0.9983. The limit of detection (LOD) is calculated to be 78.3 nM (S/N = 3). These results indicate that the proposed CDs-based nanosensor
is very useful for the detection of Hyp.
A comparison of our proposed method with some other previous reported methods for Hyp detection was studied and the results are summarized in Table S1. The LOD obtained with our developed method was lower than that reported methods [15,17,19,84-86] except the chemiluminescence method [18]. However, our proposed CDs-based nanosensor is simple, rapid and cost-effective. Moreover, the precision and accuracy of this developed method were evaluated. Ten repeated measurements of 11, 22 and 44 µM Hyp under the same conditions were performed and the results were depicted in Table S2. The recoveries and the RSD are 97.3-101.8% and 1.2-1.7%, respectively. The results indicate that our developed method have good accuracy and precision.
3.5 Possible sensing mechanism As indicated by Fig. 4A, the fluorescence intensity of the CDs decreases significantly with the increase of Hyp concentrations. Meanwhile, a red shift from 390 to 440 nm of the emission spectrum was observed. Since the surface of CDs are covered with abundant basic groups such as carboxyl and hydroxyl groups as determined by FTIR (Fig. S5) and XPS (Fig. S6), it is highly possible that the phenolic hydroxyl groups of Hyp bind to the carboxyl and hydroxyl groups of CDs through hydrogen bonding, resulting in the change in the surface of CDs. It is known that 12
CDs are good electron donors and acceptors [87]. As such, there is a possible to promote an electron transfer from the photo-excited CDs to the aromatic groups of Hyp. In another word, the CDs are wrapped by the Hyp mimicking dendrimers, allowing an effective non-radiative energy transfer from the CDs to Hyp, resulting in quenching of CDs [88].
3.6 Sample analysis The proposed method was finally used for the detection of Hyp in real samples, i.e. fufangmuji granules and human serum samples, by using the standard addition method. Table 1 summarizes the analytical results of Hyp in fufangmuji granules and human serum samples. The recoveries of Hyp in the fufangmuji granules and human serum samples are in the range of 93.3-107 % with the RSD (n = 5) are in the range of 1.16-1.72 %. The results reveal that our proposed method can be applied to detect Hyp in real samples with high precision and good repeatability.
4.0 Conclusion In this work, we developed a novel, simple, selective and sensitive method for the determination of Hyp based on the fluorescence quenching of CDs. The preparation of CDs is simple, green and economic. Compared with the reported methods for Hyp determination, our proposed method shows some advantages. First, this method is simple, economic and efficient without expensive instrument and complex operation. Second, this CDs-based fluorescence sensor has excellent sensitivity and good selectivity. Third, there is no need for further chemical modification of CDs, and all the chemicals and materials employed in this work are nontoxic or of low toxicity, making the process environmental-friendly. Furthermore, the developed method was used to detect Hyp in fufangmuji granules and human serum samples with satisfactory results 13
achieved. Our work not only adds to the database of the Hyp determination methods, but also reflects the potential application of CDs as nanosensors.
Acknowledgments The authors gratefully acknowledge the support from the National Nature Science Foundation of China (21375083).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. talanta.
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21
(A)
(B)
Fig. 1. (A) TEM image of the as-prepared CDs. (B) The particle size distribution of the as-prepared CDs.
22
(A)
(B)
Fig. 2. (A) The fluorescence intensity of CDs under different pH (1) in the absence and (2) presence of Hyp. (B) Effect of pH on the relative fluorescence intensity (Fo/F) of CDs. 0.15 mg/mL CDs and 5.5 µM Hyp were used. The error bars represent the standard deviation of the three independent measurements.
23
(A)
(B)
(C)
Fig. 3. Effects of (A) relevant metal ions, (B) biomolecules and (C) other co-existing compounds on Fo/F of CDs. 0.15 mg/mL CDs and 44 µM Hyp were used. The concentration of interfering substances are 50 µM for K+, Na+, Ca2+, Mg2+, Fe3+, Al3+, Zn2+, Cu2+, Pb2+, Ag+, Ba2+, Cr3+, Cys and Glu, 300 µM for Thr, Gly, Try, Glu, Glut, Ala, His and Asc, 100 µM for Rut, Chr, Dai, Fer, Alo, Rhe, Pue and Chl, respectively. The error bars represent the standard deviation of three independent measurements.
24
(A)
(B)
Fig. 4. (A) Fluorescence spectra of CDs in the presence of different concentrations of Hyp, from top to bottom, the concentration of Hyp are 0.00, 0.22, 1.00, 2.20, 5.50, 11.0, 16.5, 22.0, 27.5, 33.0, 38.5, 44.0, 49.5 and 55.0 µM. (B) Stern-Volmer plot of Fo/F versus Hyp concentration. 0.15 mg/mL CDs was used. The error bars represent the standard deviation of the three independent measurements.
25
Table 1 Analytical results of Hyp in real samples Sample
Fufangmuji granules
Human serum
Added (mg/g) 0.00 0.15 0.35 0.55
Found (mg/g) 0.62 0.76 0.98 1.21
Recovery (%, n = 5) ‒ 93.3 103 107
RSD (%, n = 5) 1.42 1.72 1.53 1.66
Added (mM) 0.20 0.40 0.50
Found (mM) 0.21 0.38 0.52
Recovery (%, n = 5) 105 95 104
RSD (%, n = 5) 1.37 1.46 1.28
26
Highlights
A novel sensitive and convenient method for determination of hyperin based on the fluorescence quenching of fluorescent carbon dots (CDs) was developed.
The CDs was used for determination of hyperin with a detection limit of 78.3 nM.
The CDs exhibited excellent sensitivity and selectivity to the detection of hyperin.
The method has satisfactory results for the determination of hyperin in real samples.
This work not only provides a new method for the detection of hyperin but also enriches the application of CDs.
27