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Green synthesis of carbon dots from pork and application as nanosensors for uric acid detection
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 190 (2018) 360–367
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Green synthesis of carbon dots from pork and application as nanosensors for uric acid detection Chunxi Zhao a, Yang Jiao b, Feng Hu a, Yaling Yang a,⁎ a b
Faculty of Life Science and Technology, Kunming University of Science and Technology, Yunnan Province 650500, China Civil and Environmental Engineering, Carnegie Mellon University, Pittsburgh, PA 15213-3890, United States
a r t i c l e
i n f o
Article history: Received 7 July 2017 Received in revised form 9 September 2017 Accepted 13 September 2017 Available online 15 September 2017 Keywords: Carbon dots Uric acid Pork Fluorescence quenching
a b s t r a c t In this work, a green, simple, economical method was developed in the synthesis of fluorescent carbon dots using pork as carbon source. The as-prepared carbon dots exhibit exceptional advantages including high fluorescent quantum yield (17.3%) and satisfactory chemical stability. The fluorescence of carbon dots based nanosensor can be selectively and efficiently quenched by uric acid. This phenomenon was used to develop a fluorescent method for facile detection of uric acid within a linear range of 0.1–100 μM and 100–500 μM, with a detection limit of 0.05 μM (S/N = 3). Finally, the proposed method was successfully applied in the determination of uric acid in human serum and urine samples with satisfactory recoveries, which suggested that the new nanosensors have great prospect toward the detection of uric acid in human fluids. © 2017 Published by Elsevier B.V.
1. Introduction Uric acid (UA; 2,6,8-trihydroxypurine) is the main product of purine metabolism in human bodies, which mainly exists in urine and serum [1,2]. Normal level of UA is important for body health in urine and serum range from 2.49 to 4.46 mM and 0.13 to 0.46 mM, respectively [3]. Some diseases would be caused when the level of UA is higher or lower than normal level, such as gout, kidney disease, high blood pressure, high blood lipids, atherosclerosis, Parkinson disease, Alzheimer disease and other diseases [4–6]. Therefore, it is highly necessary to monitor the level of UA in body fluids. In recent years, many methods of UA detection have been developed, including electrochemical method [7,8], enzymatic method [9], high performance liquid chromatography [10,11], chemiluminescence [12], and so on. However, these methods exist some disadvantages, such as complicated operation, time consuming and expensive cost. Fluorescence spectrophotometry has rapidly developed because it avoids the disadvantages above, and it attracts more and more attention. In recent years, nanomaterials including carbon nanomaterials, semiconductor nanomaterials, polymeric nanomaterials, metal nanomaterials have captured intensive attention and were applied in many areas, because of excellent surface effect, small size effect and macroscopic quantum tunneling effect [13–16]. Carbon dots (CDs) is a ⁎ Corresponding author. E-mail address: [email protected] (Y. Yang).
https://doi.org/10.1016/j.saa.2017.09.037 1386-1425/© 2017 Published by Elsevier B.V.
new member of carbon nanomaterial family with a size of less than 10 nm [17]. In addition to the common characteristics of nanomaterials, carbon dots also showed low cytotoxicity, excellent photostability, high biocompatibility, easy functionalization [18–20]. Therefore, CDs were widely applied in fluorescence sensors, cell imaging, metal detection, organophosphate pesticides detection [21–25] and so on. To date, many methods such as arc discharge [26], microwave digestion [27], ultrasonic oscillation [28], electrochemical method [29], hydrothermal synthesis [30] have been reported to synthesize CDs. In contrast to other methods, the hydrothermal synthesis is the most widely adopted for its simple operation, mild reaction conditions and high quantum yield. Recently, hydrothermal carbonization of aloe, chocolate, bamboo leaves, and rose-heart radish has been successfully applied to synthesize fluorescent CDs, which could be probes for tartrazine, lead ions, copper ion, iron(III) ion and cell imaging [31–34]. All of these showed that taking natural substances as carbon source for simple, economical and green synthesis of CDs is becoming one of the tendencies of CDs research. In this work, we developed a simple, low-cost and green method for synthesis of CDs from pork. Since pork is a complex that contains a number of organic and biomolecules including fat, proteins, vitamin B, vitamin C, vitamin E, carbohydrates, cholesterol and minerals, which can be useful for doping of multiple heteroatoms in the CDs without addition of any additives. In the experiment, we found that UA could quench the fluorescence of CDs, and the quenching degree was related to the concentration of UA. To our knowledge, there is few studies used CDs
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from pork for detection of UA, which provides a new method for sensitive detection of UA. In addition, this method has been successfully applied to detect UA in serum and urine samples.
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compounds during the synthesis process, so this method was more eco-friendly. 2.3. Measurement of Fluorescence Quantum Yield
2. Experimental 2.1. Materials and Apparatus Uric acid (UA) that was shown in Fig. 1, dopamine hydrochloride (DA) and urea were all purchased from Aladdin Chemistry (Shanghai, China). Oxalic acid (OA), ascorbic acid (AA), citric acid (CA) were obtained from Tianjin Zhiyuan Chemical Reagent Co., Ltd. (Tianjin, China). L-Alanine (L-Ala), L-Tyrosine (L-Tyr), DL-Leucine (DL-Leu), DLAspartic Acid (DL-Asp), L-Cystine (L-Cys), L-Glutamic acid (L-Glu) and LTryptophane (L-Try) were provided by Xinxing Chemical Reagents Co., Ltd. (Shanghai, China). NaCl, KCl, Na3PO4, Na2HPO4 were purchased from Tianjin Fengchuan Chemical Reagent Co., Ltd. (Tianjin, China). Stock solution of 1.0 mM uric acid was prepared by dissolving in 0.01 M of NaOH solution and diluted to the scale line, then stored below 4 °C. Human serum samples were kindly provided by Yanan Hospital (Kunming, China). Urine samples were provided by laboratory members. All chemicals and solvents were used without further purification. Ultra-pure water was used in each experiment. The fluorescence spectra were obtained using a G9800A Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, USA). Transmission electron microscopy (TEM) images were obtained on a FEI Tecnai G2 F30 transmission electron microscope (FEI, USA). X-ray photoelectron spectroscopy (XPS) analyses were performed on an X-ray photoelectron spectrometer (Kratos, UK). Fourier transform infrared spectra (FTIR) were recorded on a TENSOR27 FTIR spectrometer (Bruker, Germany). Absorbance measurements were performed on a UV-2550 UV–vis spectrophotometer (Shimadzu, Japan). A D8-advance X-ray diffractometer (XRD) (Bruker, Germany), a PHSJ-4A pH meter (Shanghai Instrument Electric Scientific Instrument Co., Ltd., Shanghai, China) and a vortex mixer (Hanuo Instrument Co., Ltd., XH-B, Shanghai, China) were used in the experiment.
The quantum yield (QY) of the as-synthesized CDs was measured on the basis of a procedure described previously [35]. Briefly, a solution of quinine sulfate in 0.1 M H2SO4 (QY of 54% at 360 nm, η = 1.33) was used as a standard. The value of the quantum yield was calculated according to the following equation: Q ¼ Q R ðIS =I R ÞðAR =AS Þ η2S =η2R
where Q is QY, the subscript ‘S’ refers to the samples, the subscript ‘R’ refers to quinine sulfate, A is the absorbance at the excitation wavelength, I is the integrated emission intensity and η is the solvent refraction index. 2.4. Assay Procedures Typically, 20 μL of CDs solution was diluted to 2 mL in 10 mL centrifuge tubes, then 2 mL of different concentration of UA solution or samples were added into centrifuge tubes, vortex mixed for 5 s and adjusted the pH to 8 with Na2HPO4-citric acid. After reaction for 3 min, the fluorescence intensity was recorded at an excitation wavelength of 310 nm and an emission wavelength of 412 nm. The slits for both the excitation and the emission were set to 5 nm. 2.5. Biological Sample Pretreatment The human serum samples were obtained from Yanan Hospital of Kunming and then diluted 10-fold before experiment. The urine samples were obtained from laboratory members, then decolored with a small amount of activated carbon and centrifuged to remove most of impurities, and diluted 100-fold before experiment. 3. Results and Discussion
2.2. Synthesis of CDs 3.1. Characterization of CDs CDs were prepared by a hydrothermal method using pork as carbon source. 20 g of pork was crushed and dispersed in 150 mL of deionized water, then the mixture was transferred to a Teflon-lined autoclave (200 mL) and heated at 200 °C for 10 h. After cooling down to room temperature naturally, the suspension was centrifuged at 13000 rpm for 20 min, yellow solution was obtained after removing the insoluble substances. In order to get pure CDs, the solution was filtered with a 0.22 μm membrane. Finally, the obtained CDs were stored at 4 °C for further use. The CDs could be synthesized using pork as carbon source by hydrothermal synthesis method. Compared with other synthetic methods, the hydrothermal synthesis method was simple and convenient. Furthermore, pork was used as the raw material rather than organic
Fig. 1. The molecular structures of UA.
The morphology and microstructure identified by the typical TEM image. As shown in Fig. 2A1, the CDs are nearly spherical and well separated from each other with an average diameter of about 3.5 nm. In addition, Fig. 2A2 showed that the lattice spacing is ca. 0.23 nm, corresponded to that of graphitic carbon, representing the graphitic of the CDs. As shown in Fig. 2B, X-ray diffraction (XRD) pattern of the CDs shows a broad peak at around 2θ = 23.5°, it corresponds to the graphitic structure [36]. The surface functional groups of CDs were identified by FTIR spectra and X-ray photoelectron spectroscopy (XPS). As shown in Fig. 2C, the band within the range of 3450–2950 cm−1 is attributed to \\OH and N\\H stretching vibrations. The peaks at 1672 cm− 1, 1581 cm−1, 1410 cm− 1 and 1105 cm−1 are assigned to stretching vibrations of C_O, C_C, C\\N and C\\O, respectively. The full scan XPS spectrum of CDs is shown in Fig. 2D excluded 3 peaks at 287.3, 399.9 and 532.7 eV, which separately correspond to C1s, N1s and O1s. Fig. 2E shows the C1s spectrum of CDs, the four peaks at 284.4 eV, 285.6 eV, 286.5 eV and 288.0 eV could be attributed to C_C, C\\N, C\\OH and C_O. Fig. 2F shows the N1s spectrum of CDs, the two main peaks at 399.7 eV and 400.9 eV could be linked to N\\C\\N and N\\H. Fig. 2G shows the O1s spectrum of CDs, the two main peaks at 531.2 eV and 532.3 eV could be assigned to C_O and C\\OH/C\\O\\C. The XPS results agreed with FTIR analysis. Therefore, hydrophilic groups such as \\COOH,\\NH2, and\\OH should exist on the surface of the CDs. Fig. 3A shows the UV–vis absorption and fluorescence spectra of the CDs, the as-prepared CDs exhibited a small peak at around 235 nm, and
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Fig. 2. (A1) TEM image. (A2) Size distribution of the CDs. (B) XRD pattern. (C) FTIR spectrum of the CDs. (D) Full-scan XPS spectrum of the CDs. (E) C1s XPS spectrum. (F) N1s XPS spectrum. (G) O1s XPS spectrum.
an obvious strong peak at 281 nm. The peak at 235 nm was attributed to the π–π* transitions of the C_C bond, and peak at around 281 nm was attributed to the π-π* transition of C_O bond [34,36]. Furthermore, the
maximum emission of CDs centered at 412 nm with 310 nm excitation. As shown in Fig. 3B, the excitation dependent emission spectra of the CDs. It could be observed that the emission peaks shifted to long
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Fig. 4. Stern–Volmer plots for the system of CDs-UA under temperatures of 287, 297and 307 K, respectively.
Fig. 3. (A) UV–vis absorption spectra (Abs), fluorescence excitation (Ex), and emission (Em) spectra of the CDs. Inset: Photographs of the solution of the CDs taken under visible light (left) and 254 nm UV light (right). (B) Fluorescence emission spectra of CDs at different excitation wavelengths from 270 to 330 nm in 10 nm increments.
wavelength when the excitation wavelength varied from 270 to 330 nm in 10 nm increments. 3.2. Possible Quenching Mechanism The quenching of fluorescence by UA can be described by the Stern– Volmer equation: F0 =F ¼ 1 þ KSV C ¼ 1 þ Kq τ0 C where KSV and Kq are the Stern–Volmer quenching constant and the bimolecular quenching constant, respectively. C is the concentration of UA. τ0 is the average lifetime of the C-dots without any other fluorescence quencher, with a general value of 10−8 s [31]. F0 and F are the fluorescence intensity of CDs without UA and with different concentration of UA, respectively. Generally, the static fluorescence quenching constants will decrease with the rise of temperature, while the dynamic fluorescence quenching constants will increase with the rise of the system temperature. Fig. 4 shows the change of fluorescence intensities of the CDs at 287, 297, 307 K. The values of KSV and Kq were shown in Table 1. As the results shown, with the rise of the system temperature, the fluorescence quenching constants decreased. Additionally, the values of Kq are all larger than 2.0 × 1010 L mol−1 s−1, which is the maximum dynamic quenching constant [31]. Therefore, the fluorescence quenching of CDs by UA may be a static quenching process. In this work, the fluorescence intensity of CDs decreased with the addition of UA, this phenomenon may be result from the formation of a ground-
state complex and the aggregation of CDs. The complex was formed though the molecular interactions including electrostatic and hydrogen bonding interactions. FTIR and XPS have confirmed that the existence of \\COOH,\\OH and\\NH2 on the surface of CDs, the carboxyl group has a negative charge under weakly alkaline condition while secondary amine group in the UA molecule has a positive charge, there is an electrostatic interaction between them. Simultaneously, hydrogen bond can be formed between the oxygen atom of carbonyl group in the uric acid molecule and the hydrogen atom as well as nitrogen atom of the amino group on the surface of the CDs. Moreover, some researchers [37] have revealed that after the addition of UA to the QDs solution, the surface of QDs might be changed, which would result in poor luminescence efficiency. The changes on the surface may cause the aggregation of QDs, and then the fluorescence of QDs was quenched. The UA would better dispersed in weakly alkaline conditions, which promote the maximal response of the CDs to UA. With the addition of UA, the surface of CDs may be changed and lead to the aggregation of CDs, then fluorescence was quenched. Therefore, the fluorescence quenching may be the result of the combination of the above two reasons. The most possible quenching mechanism of fluorescence intensity of the prepared CDs is surface absorption (Scheme 1). 3.3. Fluorescence Response Strategy for the Detection of UA 3.3.1. Effect of pH Value pH may not only affect the fluorescence intensity of CDs, but also affect fluorescence quenching of uric acid to the CDs. Therefore, the effect of pH from 3.0 to 11.0 on experimental system was investigated. As shown in Fig. 5A, the fluorescence intensity of the CDs was found to be pH dependent, and the fluorescence had no significant change in the pH range of 3.0–8.0 but decreased above pH 8.0. Simultaneously, Fig. 5B shown that fluorescence quenching was enhanced in the pH range of 3.0–8.0, then remained essentially stable above pH 8.0. That may because UA exhibits weak acidity, which cannot dispersed well in acid condition, and result in bad response to CDs. The results show
Table 1 Stern–Volmer quenching constants for the interaction of CDs and UA at different temperatures. pH 8 8 8
T (K) 287 297 307
KSV (L mol−1) 3
3.6 × 10 3.5 × 103 3.2 × 103
Kq (L mol−1 s−1) 11
3.6 × 10 3.5 × 1011 3.2 × 1011
R2
RSD (%)
0.9910 0.9922 0.9917
2.8 3.5 3.1
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Scheme 1. Scheme of the synthetic strategy for the CDs and the principle of fluorescence quenching.
that the maximal response of the system was obtained at pH 8.0, and this pH was selected as the optimum pH for the buffer solution. 3.3.2. Fluorescence Stability Investigation To investigate the stability of CDs for the detection of UA, the fluorescence intensity of CDs in the presence of UA was investigated in different reaction time. As shown in Fig. 6, the fluorescence intensity of CDs was quenched after the addition of UA, and the stable fluorescence intensity was obtained after 3 min reaction. In addition, the fluorescence intensity of experimental system remained constant when the reaction time was prolonged more than 30 min. These results showed that this method was fast and stable for the detection of UA. Therefore, the reaction time of 3 min was selected for the UA determination. In addition, as shown in Supplementary materials (Fig. S1), the as-prepared CDs had good long stability. The CDs still own strong fluorescence intensity after 4 months storage at 4 °C. Simultaneously, CDs solution can be dried in vacuum oven to obtain powders, which could be redispersed in water for using. The fluorescence intensity had no observable decrease.
Fig. 5. (A) The effect of pH on the fluorescence intensity of CDs; (B) the effect of pH on fluorescence quenching of UA (250 μM) to the CDs.
Fig. 6. Fluorescence intensity of CDs with UA (250 μM) at different reaction time.
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3.3.3. The Sensitivity of UA Detection As shown in Fig. 7A, with the addition of various concentration of UA, the fluorescence intensity of CDs decreased gradually. In Fig. 7B
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and C, the ratio between the fluorescence intensity of the system after and before adding UA linearly decreased with UA concentration from 0.1 to 100 μM with a regression equation of F/F0 = − 0.0027C + 0.9954 (r2 = 0.9979), and from 100 to 500 μM with a regression equation of F/F0 = −0.0011C + 0.8088 (r2 = 0.9924), where F and F0 are the fluorescent intensities of CDs in the presence and absence of UA, and C is the concentration of UA. The detection limit of UA is 0.05 μM, which was obtained at a signal-to-noise ratio of 3. Compared with other reported literatures which detected UA, the CDs proposed in this work gives a relatively lower limit of detection (Table 2). 3.3.4. Selectivity The selectivity was examined by monitoring the change of fluorescence intensity of CDs in the presence of different possible interfering substances, including urea, AA, CA, DA, OA, L-Ala, L-Tyr, DL-Leu, DL-Asp, L-Cys, L-Glu, L-Try, NaCl, KCl, Na3PO4. As shown in Fig. 8A, only UA could efficiently quenched the fluorescence intensity of the CDs, while the quenching effect of the other possible interfering substances was negligible. Interestingly, as shown in Fig. 8B, it could be note that only the fluorescence intensity of the CDs that synthesized using pork as a carbon source was quenched by uric acid, but the fluorescence intensity of the CDs synthesized using chicken and beef as carbon sources cannot be quenched. This result revealed that there may be a certain strict quenching relationship between the CDs synthesized with pork and uric acid, it deserves our further study. Therefore, high selectivity existed between UA and the as-prepared CDs, the CDs could be used for the detection of UA.
3.3.5. Analysis of UA in Real Samples To assess the practicality of the method, UA in the human serum and urine were detected by this method. Table 2 showed the quantification results of the uric acid in human serum samples and urine samples. The relative standard deviation (RSD) of each sample is below 6%. Furthermore, in order to test the accuracy of the proposed method, the UA concentrations of the same samples were analyzed simultaneously by the HPLC method. The results are shown in Table 3 and Supplementary materials (Fig. S2), a t-test at the 95% confidence level revealed that the UA concentrations found had no significant difference between these two methods. These results confirmed that the prepared CDs could be potentially applicable for the measurement of UA concentration in human serum and urine. 4. Conclusions In conclusion, we developed a green and low cost synthesis route to prepare highly fluorescent CDs from pork, and the fluorescence intensity of CDs could be quenched by UA, so the CDs were used as nanosensors for the sensitive and selective detection of UA in human fluids. This novel proposed method offered good linear UA detection range of 0.1–100 μM and 100–500 μM, with a detection limit of 0.05 μM. The practical application of the CDs was successfully demonstrated by determining the concentration of UA in human serum and urine samples. In addition, compared with those fluorescence methods based on semiconductor quantum dots, the proposed strategy is much more environmentally friendly. Table 2 Comparison of different methods for the determination of UA.
Fig. 7. (A) The relationship between the fluorescence intensity quenching and the various UA concentration (0.1–500 μM). Inset: Emission spectra of the CDs in the presence of various concentrations (0.1–500 μM) of UA in Na2HPO4-citric acid (pH 8.00) buffer solution. (B, C) The linear relationship between fluorescence quenching of the CDs and the UA concentration in the range from 0.1 to 100 μM (B), 100 to 500 μM (C).
Analytical method
Linear range (μM)
LOD (μM)
Ref.
Electrochemical method Fluorescent sensor Fluorescent sensor Electrochemical method Electrochemical method Fluorescent sensor
0.1–30 125–1000 0.22–6 2–30 0.3–100 0.1–100, 100–500
0.142 125 0.1 0.85 0.3 0.05
[38] [3] [39] [40] [41] This work
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References
Fig. 8. (A) Selectivity of the CDs to different possible interfering substances with the concentration of 500 μM; (B) the effect of different concentration of UA on the CDs synthesized with beef, chicken and pork.
Acknowledgments The work was strongly supported by the Analysis and Testing Foundation of Kunming University of Science and Technology (2017M20162118092). Appendix A. Supplementary Data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.saa.2017.09.037. Table 3 Determination of UA in real samples (n = 3). Samples
Human serum Urine
1 2 3 1 2 3
Fluorescence detected (mM)
RSD HPLC (%, n = 3) detected (mM)
RSD (%, n = 3)
0.32 0.17 0.4 1.93 4.14 2.67
2.6 5.6 4.4 3.6 3.5 4.6
1.8 2.6 2.4 3.4 3.1 2.1
0.29 0.19 0.37 2.17 4.02 2.78
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Green synthesis of carbon dots from pork and application as nanosensors for uric acid detection Chunxi Zhao a, Yang Jiao b, Feng Hu a, Yaling Yang a,⁎ a b
Faculty of Life Science and Technology, Kunming University of Science and Technology, Yunnan Province 650500, China Civil and Environmental Engineering, Carnegie Mellon University, Pittsburgh, PA 15213-3890, United States
a r t i c l e
i n f o
Article history: Received 7 July 2017 Received in revised form 9 September 2017 Accepted 13 September 2017 Available online 15 September 2017 Keywords: Carbon dots Uric acid Pork Fluorescence quenching
a b s t r a c t In this work, a green, simple, economical method was developed in the synthesis of fluorescent carbon dots using pork as carbon source. The as-prepared carbon dots exhibit exceptional advantages including high fluorescent quantum yield (17.3%) and satisfactory chemical stability. The fluorescence of carbon dots based nanosensor can be selectively and efficiently quenched by uric acid. This phenomenon was used to develop a fluorescent method for facile detection of uric acid within a linear range of 0.1–100 μM and 100–500 μM, with a detection limit of 0.05 μM (S/N = 3). Finally, the proposed method was successfully applied in the determination of uric acid in human serum and urine samples with satisfactory recoveries, which suggested that the new nanosensors have great prospect toward the detection of uric acid in human fluids. © 2017 Published by Elsevier B.V.
1. Introduction Uric acid (UA; 2,6,8-trihydroxypurine) is the main product of purine metabolism in human bodies, which mainly exists in urine and serum [1,2]. Normal level of UA is important for body health in urine and serum range from 2.49 to 4.46 mM and 0.13 to 0.46 mM, respectively [3]. Some diseases would be caused when the level of UA is higher or lower than normal level, such as gout, kidney disease, high blood pressure, high blood lipids, atherosclerosis, Parkinson disease, Alzheimer disease and other diseases [4–6]. Therefore, it is highly necessary to monitor the level of UA in body fluids. In recent years, many methods of UA detection have been developed, including electrochemical method [7,8], enzymatic method [9], high performance liquid chromatography [10,11], chemiluminescence [12], and so on. However, these methods exist some disadvantages, such as complicated operation, time consuming and expensive cost. Fluorescence spectrophotometry has rapidly developed because it avoids the disadvantages above, and it attracts more and more attention. In recent years, nanomaterials including carbon nanomaterials, semiconductor nanomaterials, polymeric nanomaterials, metal nanomaterials have captured intensive attention and were applied in many areas, because of excellent surface effect, small size effect and macroscopic quantum tunneling effect [13–16]. Carbon dots (CDs) is a ⁎ Corresponding author. E-mail address: [email protected] (Y. Yang).
https://doi.org/10.1016/j.saa.2017.09.037 1386-1425/© 2017 Published by Elsevier B.V.
new member of carbon nanomaterial family with a size of less than 10 nm [17]. In addition to the common characteristics of nanomaterials, carbon dots also showed low cytotoxicity, excellent photostability, high biocompatibility, easy functionalization [18–20]. Therefore, CDs were widely applied in fluorescence sensors, cell imaging, metal detection, organophosphate pesticides detection [21–25] and so on. To date, many methods such as arc discharge [26], microwave digestion [27], ultrasonic oscillation [28], electrochemical method [29], hydrothermal synthesis [30] have been reported to synthesize CDs. In contrast to other methods, the hydrothermal synthesis is the most widely adopted for its simple operation, mild reaction conditions and high quantum yield. Recently, hydrothermal carbonization of aloe, chocolate, bamboo leaves, and rose-heart radish has been successfully applied to synthesize fluorescent CDs, which could be probes for tartrazine, lead ions, copper ion, iron(III) ion and cell imaging [31–34]. All of these showed that taking natural substances as carbon source for simple, economical and green synthesis of CDs is becoming one of the tendencies of CDs research. In this work, we developed a simple, low-cost and green method for synthesis of CDs from pork. Since pork is a complex that contains a number of organic and biomolecules including fat, proteins, vitamin B, vitamin C, vitamin E, carbohydrates, cholesterol and minerals, which can be useful for doping of multiple heteroatoms in the CDs without addition of any additives. In the experiment, we found that UA could quench the fluorescence of CDs, and the quenching degree was related to the concentration of UA. To our knowledge, there is few studies used CDs
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from pork for detection of UA, which provides a new method for sensitive detection of UA. In addition, this method has been successfully applied to detect UA in serum and urine samples.
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compounds during the synthesis process, so this method was more eco-friendly. 2.3. Measurement of Fluorescence Quantum Yield
2. Experimental 2.1. Materials and Apparatus Uric acid (UA) that was shown in Fig. 1, dopamine hydrochloride (DA) and urea were all purchased from Aladdin Chemistry (Shanghai, China). Oxalic acid (OA), ascorbic acid (AA), citric acid (CA) were obtained from Tianjin Zhiyuan Chemical Reagent Co., Ltd. (Tianjin, China). L-Alanine (L-Ala), L-Tyrosine (L-Tyr), DL-Leucine (DL-Leu), DLAspartic Acid (DL-Asp), L-Cystine (L-Cys), L-Glutamic acid (L-Glu) and LTryptophane (L-Try) were provided by Xinxing Chemical Reagents Co., Ltd. (Shanghai, China). NaCl, KCl, Na3PO4, Na2HPO4 were purchased from Tianjin Fengchuan Chemical Reagent Co., Ltd. (Tianjin, China). Stock solution of 1.0 mM uric acid was prepared by dissolving in 0.01 M of NaOH solution and diluted to the scale line, then stored below 4 °C. Human serum samples were kindly provided by Yanan Hospital (Kunming, China). Urine samples were provided by laboratory members. All chemicals and solvents were used without further purification. Ultra-pure water was used in each experiment. The fluorescence spectra were obtained using a G9800A Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, USA). Transmission electron microscopy (TEM) images were obtained on a FEI Tecnai G2 F30 transmission electron microscope (FEI, USA). X-ray photoelectron spectroscopy (XPS) analyses were performed on an X-ray photoelectron spectrometer (Kratos, UK). Fourier transform infrared spectra (FTIR) were recorded on a TENSOR27 FTIR spectrometer (Bruker, Germany). Absorbance measurements were performed on a UV-2550 UV–vis spectrophotometer (Shimadzu, Japan). A D8-advance X-ray diffractometer (XRD) (Bruker, Germany), a PHSJ-4A pH meter (Shanghai Instrument Electric Scientific Instrument Co., Ltd., Shanghai, China) and a vortex mixer (Hanuo Instrument Co., Ltd., XH-B, Shanghai, China) were used in the experiment.
The quantum yield (QY) of the as-synthesized CDs was measured on the basis of a procedure described previously [35]. Briefly, a solution of quinine sulfate in 0.1 M H2SO4 (QY of 54% at 360 nm, η = 1.33) was used as a standard. The value of the quantum yield was calculated according to the following equation: Q ¼ Q R ðIS =I R ÞðAR =AS Þ η2S =η2R
where Q is QY, the subscript ‘S’ refers to the samples, the subscript ‘R’ refers to quinine sulfate, A is the absorbance at the excitation wavelength, I is the integrated emission intensity and η is the solvent refraction index. 2.4. Assay Procedures Typically, 20 μL of CDs solution was diluted to 2 mL in 10 mL centrifuge tubes, then 2 mL of different concentration of UA solution or samples were added into centrifuge tubes, vortex mixed for 5 s and adjusted the pH to 8 with Na2HPO4-citric acid. After reaction for 3 min, the fluorescence intensity was recorded at an excitation wavelength of 310 nm and an emission wavelength of 412 nm. The slits for both the excitation and the emission were set to 5 nm. 2.5. Biological Sample Pretreatment The human serum samples were obtained from Yanan Hospital of Kunming and then diluted 10-fold before experiment. The urine samples were obtained from laboratory members, then decolored with a small amount of activated carbon and centrifuged to remove most of impurities, and diluted 100-fold before experiment. 3. Results and Discussion
2.2. Synthesis of CDs 3.1. Characterization of CDs CDs were prepared by a hydrothermal method using pork as carbon source. 20 g of pork was crushed and dispersed in 150 mL of deionized water, then the mixture was transferred to a Teflon-lined autoclave (200 mL) and heated at 200 °C for 10 h. After cooling down to room temperature naturally, the suspension was centrifuged at 13000 rpm for 20 min, yellow solution was obtained after removing the insoluble substances. In order to get pure CDs, the solution was filtered with a 0.22 μm membrane. Finally, the obtained CDs were stored at 4 °C for further use. The CDs could be synthesized using pork as carbon source by hydrothermal synthesis method. Compared with other synthetic methods, the hydrothermal synthesis method was simple and convenient. Furthermore, pork was used as the raw material rather than organic
Fig. 1. The molecular structures of UA.
The morphology and microstructure identified by the typical TEM image. As shown in Fig. 2A1, the CDs are nearly spherical and well separated from each other with an average diameter of about 3.5 nm. In addition, Fig. 2A2 showed that the lattice spacing is ca. 0.23 nm, corresponded to that of graphitic carbon, representing the graphitic of the CDs. As shown in Fig. 2B, X-ray diffraction (XRD) pattern of the CDs shows a broad peak at around 2θ = 23.5°, it corresponds to the graphitic structure [36]. The surface functional groups of CDs were identified by FTIR spectra and X-ray photoelectron spectroscopy (XPS). As shown in Fig. 2C, the band within the range of 3450–2950 cm−1 is attributed to \\OH and N\\H stretching vibrations. The peaks at 1672 cm− 1, 1581 cm−1, 1410 cm− 1 and 1105 cm−1 are assigned to stretching vibrations of C_O, C_C, C\\N and C\\O, respectively. The full scan XPS spectrum of CDs is shown in Fig. 2D excluded 3 peaks at 287.3, 399.9 and 532.7 eV, which separately correspond to C1s, N1s and O1s. Fig. 2E shows the C1s spectrum of CDs, the four peaks at 284.4 eV, 285.6 eV, 286.5 eV and 288.0 eV could be attributed to C_C, C\\N, C\\OH and C_O. Fig. 2F shows the N1s spectrum of CDs, the two main peaks at 399.7 eV and 400.9 eV could be linked to N\\C\\N and N\\H. Fig. 2G shows the O1s spectrum of CDs, the two main peaks at 531.2 eV and 532.3 eV could be assigned to C_O and C\\OH/C\\O\\C. The XPS results agreed with FTIR analysis. Therefore, hydrophilic groups such as \\COOH,\\NH2, and\\OH should exist on the surface of the CDs. Fig. 3A shows the UV–vis absorption and fluorescence spectra of the CDs, the as-prepared CDs exhibited a small peak at around 235 nm, and
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Fig. 2. (A1) TEM image. (A2) Size distribution of the CDs. (B) XRD pattern. (C) FTIR spectrum of the CDs. (D) Full-scan XPS spectrum of the CDs. (E) C1s XPS spectrum. (F) N1s XPS spectrum. (G) O1s XPS spectrum.
an obvious strong peak at 281 nm. The peak at 235 nm was attributed to the π–π* transitions of the C_C bond, and peak at around 281 nm was attributed to the π-π* transition of C_O bond [34,36]. Furthermore, the
maximum emission of CDs centered at 412 nm with 310 nm excitation. As shown in Fig. 3B, the excitation dependent emission spectra of the CDs. It could be observed that the emission peaks shifted to long
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Fig. 4. Stern–Volmer plots for the system of CDs-UA under temperatures of 287, 297and 307 K, respectively.
Fig. 3. (A) UV–vis absorption spectra (Abs), fluorescence excitation (Ex), and emission (Em) spectra of the CDs. Inset: Photographs of the solution of the CDs taken under visible light (left) and 254 nm UV light (right). (B) Fluorescence emission spectra of CDs at different excitation wavelengths from 270 to 330 nm in 10 nm increments.
wavelength when the excitation wavelength varied from 270 to 330 nm in 10 nm increments. 3.2. Possible Quenching Mechanism The quenching of fluorescence by UA can be described by the Stern– Volmer equation: F0 =F ¼ 1 þ KSV C ¼ 1 þ Kq τ0 C where KSV and Kq are the Stern–Volmer quenching constant and the bimolecular quenching constant, respectively. C is the concentration of UA. τ0 is the average lifetime of the C-dots without any other fluorescence quencher, with a general value of 10−8 s [31]. F0 and F are the fluorescence intensity of CDs without UA and with different concentration of UA, respectively. Generally, the static fluorescence quenching constants will decrease with the rise of temperature, while the dynamic fluorescence quenching constants will increase with the rise of the system temperature. Fig. 4 shows the change of fluorescence intensities of the CDs at 287, 297, 307 K. The values of KSV and Kq were shown in Table 1. As the results shown, with the rise of the system temperature, the fluorescence quenching constants decreased. Additionally, the values of Kq are all larger than 2.0 × 1010 L mol−1 s−1, which is the maximum dynamic quenching constant [31]. Therefore, the fluorescence quenching of CDs by UA may be a static quenching process. In this work, the fluorescence intensity of CDs decreased with the addition of UA, this phenomenon may be result from the formation of a ground-
state complex and the aggregation of CDs. The complex was formed though the molecular interactions including electrostatic and hydrogen bonding interactions. FTIR and XPS have confirmed that the existence of \\COOH,\\OH and\\NH2 on the surface of CDs, the carboxyl group has a negative charge under weakly alkaline condition while secondary amine group in the UA molecule has a positive charge, there is an electrostatic interaction between them. Simultaneously, hydrogen bond can be formed between the oxygen atom of carbonyl group in the uric acid molecule and the hydrogen atom as well as nitrogen atom of the amino group on the surface of the CDs. Moreover, some researchers [37] have revealed that after the addition of UA to the QDs solution, the surface of QDs might be changed, which would result in poor luminescence efficiency. The changes on the surface may cause the aggregation of QDs, and then the fluorescence of QDs was quenched. The UA would better dispersed in weakly alkaline conditions, which promote the maximal response of the CDs to UA. With the addition of UA, the surface of CDs may be changed and lead to the aggregation of CDs, then fluorescence was quenched. Therefore, the fluorescence quenching may be the result of the combination of the above two reasons. The most possible quenching mechanism of fluorescence intensity of the prepared CDs is surface absorption (Scheme 1). 3.3. Fluorescence Response Strategy for the Detection of UA 3.3.1. Effect of pH Value pH may not only affect the fluorescence intensity of CDs, but also affect fluorescence quenching of uric acid to the CDs. Therefore, the effect of pH from 3.0 to 11.0 on experimental system was investigated. As shown in Fig. 5A, the fluorescence intensity of the CDs was found to be pH dependent, and the fluorescence had no significant change in the pH range of 3.0–8.0 but decreased above pH 8.0. Simultaneously, Fig. 5B shown that fluorescence quenching was enhanced in the pH range of 3.0–8.0, then remained essentially stable above pH 8.0. That may because UA exhibits weak acidity, which cannot dispersed well in acid condition, and result in bad response to CDs. The results show
Table 1 Stern–Volmer quenching constants for the interaction of CDs and UA at different temperatures. pH 8 8 8
T (K) 287 297 307
KSV (L mol−1) 3
3.6 × 10 3.5 × 103 3.2 × 103
Kq (L mol−1 s−1) 11
3.6 × 10 3.5 × 1011 3.2 × 1011
R2
RSD (%)
0.9910 0.9922 0.9917
2.8 3.5 3.1
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Scheme 1. Scheme of the synthetic strategy for the CDs and the principle of fluorescence quenching.
that the maximal response of the system was obtained at pH 8.0, and this pH was selected as the optimum pH for the buffer solution. 3.3.2. Fluorescence Stability Investigation To investigate the stability of CDs for the detection of UA, the fluorescence intensity of CDs in the presence of UA was investigated in different reaction time. As shown in Fig. 6, the fluorescence intensity of CDs was quenched after the addition of UA, and the stable fluorescence intensity was obtained after 3 min reaction. In addition, the fluorescence intensity of experimental system remained constant when the reaction time was prolonged more than 30 min. These results showed that this method was fast and stable for the detection of UA. Therefore, the reaction time of 3 min was selected for the UA determination. In addition, as shown in Supplementary materials (Fig. S1), the as-prepared CDs had good long stability. The CDs still own strong fluorescence intensity after 4 months storage at 4 °C. Simultaneously, CDs solution can be dried in vacuum oven to obtain powders, which could be redispersed in water for using. The fluorescence intensity had no observable decrease.
Fig. 5. (A) The effect of pH on the fluorescence intensity of CDs; (B) the effect of pH on fluorescence quenching of UA (250 μM) to the CDs.
Fig. 6. Fluorescence intensity of CDs with UA (250 μM) at different reaction time.
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3.3.3. The Sensitivity of UA Detection As shown in Fig. 7A, with the addition of various concentration of UA, the fluorescence intensity of CDs decreased gradually. In Fig. 7B
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and C, the ratio between the fluorescence intensity of the system after and before adding UA linearly decreased with UA concentration from 0.1 to 100 μM with a regression equation of F/F0 = − 0.0027C + 0.9954 (r2 = 0.9979), and from 100 to 500 μM with a regression equation of F/F0 = −0.0011C + 0.8088 (r2 = 0.9924), where F and F0 are the fluorescent intensities of CDs in the presence and absence of UA, and C is the concentration of UA. The detection limit of UA is 0.05 μM, which was obtained at a signal-to-noise ratio of 3. Compared with other reported literatures which detected UA, the CDs proposed in this work gives a relatively lower limit of detection (Table 2). 3.3.4. Selectivity The selectivity was examined by monitoring the change of fluorescence intensity of CDs in the presence of different possible interfering substances, including urea, AA, CA, DA, OA, L-Ala, L-Tyr, DL-Leu, DL-Asp, L-Cys, L-Glu, L-Try, NaCl, KCl, Na3PO4. As shown in Fig. 8A, only UA could efficiently quenched the fluorescence intensity of the CDs, while the quenching effect of the other possible interfering substances was negligible. Interestingly, as shown in Fig. 8B, it could be note that only the fluorescence intensity of the CDs that synthesized using pork as a carbon source was quenched by uric acid, but the fluorescence intensity of the CDs synthesized using chicken and beef as carbon sources cannot be quenched. This result revealed that there may be a certain strict quenching relationship between the CDs synthesized with pork and uric acid, it deserves our further study. Therefore, high selectivity existed between UA and the as-prepared CDs, the CDs could be used for the detection of UA.
3.3.5. Analysis of UA in Real Samples To assess the practicality of the method, UA in the human serum and urine were detected by this method. Table 2 showed the quantification results of the uric acid in human serum samples and urine samples. The relative standard deviation (RSD) of each sample is below 6%. Furthermore, in order to test the accuracy of the proposed method, the UA concentrations of the same samples were analyzed simultaneously by the HPLC method. The results are shown in Table 3 and Supplementary materials (Fig. S2), a t-test at the 95% confidence level revealed that the UA concentrations found had no significant difference between these two methods. These results confirmed that the prepared CDs could be potentially applicable for the measurement of UA concentration in human serum and urine. 4. Conclusions In conclusion, we developed a green and low cost synthesis route to prepare highly fluorescent CDs from pork, and the fluorescence intensity of CDs could be quenched by UA, so the CDs were used as nanosensors for the sensitive and selective detection of UA in human fluids. This novel proposed method offered good linear UA detection range of 0.1–100 μM and 100–500 μM, with a detection limit of 0.05 μM. The practical application of the CDs was successfully demonstrated by determining the concentration of UA in human serum and urine samples. In addition, compared with those fluorescence methods based on semiconductor quantum dots, the proposed strategy is much more environmentally friendly. Table 2 Comparison of different methods for the determination of UA.
Fig. 7. (A) The relationship between the fluorescence intensity quenching and the various UA concentration (0.1–500 μM). Inset: Emission spectra of the CDs in the presence of various concentrations (0.1–500 μM) of UA in Na2HPO4-citric acid (pH 8.00) buffer solution. (B, C) The linear relationship between fluorescence quenching of the CDs and the UA concentration in the range from 0.1 to 100 μM (B), 100 to 500 μM (C).
Analytical method
Linear range (μM)
LOD (μM)
Ref.
Electrochemical method Fluorescent sensor Fluorescent sensor Electrochemical method Electrochemical method Fluorescent sensor
0.1–30 125–1000 0.22–6 2–30 0.3–100 0.1–100, 100–500
0.142 125 0.1 0.85 0.3 0.05
[38] [3] [39] [40] [41] This work
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References
Fig. 8. (A) Selectivity of the CDs to different possible interfering substances with the concentration of 500 μM; (B) the effect of different concentration of UA on the CDs synthesized with beef, chicken and pork.
Acknowledgments The work was strongly supported by the Analysis and Testing Foundation of Kunming University of Science and Technology (2017M20162118092). Appendix A. Supplementary Data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.saa.2017.09.037. Table 3 Determination of UA in real samples (n = 3). Samples
Human serum Urine
1 2 3 1 2 3
Fluorescence detected (mM)
RSD HPLC (%, n = 3) detected (mM)
RSD (%, n = 3)
0.32 0.17 0.4 1.93 4.14 2.67
2.6 5.6 4.4 3.6 3.5 4.6
1.8 2.6 2.4 3.4 3.1 2.1
0.29 0.19 0.37 2.17 4.02 2.78
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