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Hot-injection strategy for 1-min synthesis of carbon dots from oxygen-containing organic solvents: Toward fluorescence sensing of hemoglobin
Dyes and Pigments 165 (2019) 429–435
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Hot-injection strategy for 1-min synthesis of carbon dots from oxygencontaining organic solvents: Toward fluorescence sensing of hemoglobin
T
Yaoping Hua,∗, Zhijin Gaob a b
School of Materials Science and Chemical Engineering, Ningbo University, Ningbo, 315211, China Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo, 315201, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Hot-injection One-minute synthesis Carbon dots Oxygen-containing organic solvents Sensing of hemoglobin
Here, we present a rapid and general fabrication methodology for fluorescent carbon dots (CDs) via injecting various oxygen-containing organic solvents including acetone, ethanol, ethyl acetate, tetrahydrofuran, and diethyl ether into hot concentrated sulfuric acid. All these small organic molecules are recognized as common, cheap, and abundant carbon precursors, which can convert into CDs just in 1 min through protonation, polymerization and aromatization. The as-prepared CDs with disordered amorphous cores and plentiful oxygenous groups show fascinating photoluminescence features, that can be used for sensitive and selective detection of hemoglobin (Hb) based on fluorescence quenching. The linear detection range is estimated to be 2–360 nM and the limit of detection is as low as 0.79 nM. After careful investigations, the inner filter effect is proposed to explain the sensing mechanism because the absorption band of Hb fully covers the emission spectrum of CDs and the fluorescence lifetime of CDs remains nearly constant after addition of Hb. The sensor is successfully applied for Hb detection in human blood samples, exhibiting a great potential for quantitative analysis of Hb in complex biological matrices.
1. Introduction Carbon dots (CDs) represent a new family of fluorescent carbogenic materials, generally containing small aromatic carbon clusters in the core and being functionalized with abundant oxygenous groups at the surface [1,2]. Taking advantages of alluring optical properties, low toxicity, robust chemical inertness, and facile modification [3–6], CDs provide unprecedented opportunities for applications in bioimaging, sensors, nanomedicine, photocatalysis, optoelectronics, etc [7–15]. Since their accident discovery as byproducts during electrophoretic purification of single-walled carbon nanotubes [16], numerous approaches including laser ablation [17], electrochemical exfoliation [18–20], acidic oxidation [21,22], thermal oxidation [23,24], ultrasonic treatment [25,26], microwave heating [27–30], and hydrothermal technique [31–35], have been explored to fabricate CDs using a broad range of carbon feedstocks. However, except microwave method, other above-mentioned approaches usually suffer from a long reaction time that lasts up to several hours or even a few days. In addition, some synthetic approaches require special instruments, or uncommon reagents, or complicated procedures, which restrict their widespread usage. Currently, despite many impressive advances, the rapid synthesis of CDs from cheap carbon sources with simple apparatus is still actively
∗
pursued. Hemoglobin (Hb), containing heme groups, is a metalloprotein in the red blood cells of vertebrates. It transports oxygen from the lungs to the rest of the body to permit aerobic respiration. The deficiency of Hb decreases blood oxygen-carrying capacity, causing symptoms of anemia, while the high levels of Hb may result in lung disease and certain tumors [36,37]. Therefore, the accurate determination of the trace amount of Hb in biosystems is of great significance for medical diagnosis to control the related clinical illness. Unfortunately, the recently reported methods for Hb detection like colorimetric approaches [38], spectrophotometric assays [36,39,40], electrochemical measurements [37,41], and chemiluminescence determination [42] have some drawbacks such as the requirement of expensive materials, the need of tedious sample pretreatment, and relatively high detection limit. At present, it is highly demanded to develop feasible methods for simple, cost-effective, and sensitive detection of Hb in complex biological media. Herein, we report a novel strategy for rapid synthesis of fluorescent CDs by injecting various oxygen-containing organic solvents into hot concentrated sulfuric acid (Scheme 1). The selected solvents including acetone, ethanol, ethyl acetate, tetrahydrofuran, and diethyl ether, serve as common, cheap, and abundant precursors, all of which can
Corresponding author. E-mail address: [email protected] (Y. Hu).
https://doi.org/10.1016/j.dyepig.2019.03.001 Received 1 February 2019; Received in revised form 26 February 2019; Accepted 1 March 2019 Available online 05 March 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
Dyes and Pigments 165 (2019) 429–435
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Scheme 1. A diagram about the rapid synthesis of CDs from oxygen-containing organic solvents using hot-injection strategy, and the application of CDs for Hb sensing.
whole blood samples were first diluted 100,000-fold in deionized water, and then sonicated for 30 s to release Hb from erythrocytes. After centrifugation at 3000 rpm for 10 min, the as-prepared blood sample (4000 μL) was mixed with the standard Hb solution in a certain concentration (500 μL) and the CD dispersion (500 μL). The fluorescence measurement was performed as described above. All the tests were conducted in triplicate.
efficiently convert into CDs in 1 min without need of any special equipment. The resultant acetone-derived CDs with partially disordered carbon structures exhibit a highly selective and sensitive detection of Hb through a fluorescence quenching process. After detailed investigations, the inner filter effect is proposed to elucidate the “on-off” sensing mechanism. Moreover, the fluorescence sensor has been successfully applied for the assay of Hb in human blood samples, and satisfactory results are achieved.
3. Results and discussion
2. Experimental
3.1. Morphology, structure and composition of CDs
2.1. Chemicals
As a sole carbon source, acetone (1 mL) was rapidly injected into the hot concentrated sulfuric acid (2 mL) at 170 °C, and the reaction at that temperature lasted for only 1 min, immediately producing a dark brown dispersion of CDs. The TEM image (Fig. 1a) shows that the as-synthesized CDs are nearly spherical and well separated from each other. They have a narrow size distribution ranging from 2.0 to 5.0 nm with the maximum population of approximately 3.5 nm (Fig. 1b). The high-resolution TEM image (Fig. 1a, inset) reveals the amorphous nature of CDs without clear lattice fringes. The XRD pattern (Fig. 1c) displays two broad diffraction peaks at 2θ angles of about 23.8° and 42.7°, which are attributable to amorphous carbon composed of aromatic carbon clusters oriented in a considerably random fashion [44]. In the Raman spectrum (Fig. 1d), the D band at 1358 cm−1 stands for the sp3 carbon species, and the G band at 1579 cm−1 represents the sp2 carbon networks. The coexistence of D and G bands with intensity ratio of 0.71 reflects some disorder or defects in the carbon matrix [45]. The elemental compositions and chemical bonds of CDs were investigated by the XPS measurement. The survey spectrum (Fig. 2a) shows sharp C1s, O1s and weak S2s, S2p signals. The atomic ratios of C, O, and S are 67.4%, 29.0%, and 3.6% respectively, demonstrating that the sample comprises mainly carbon and oxygen, as well as a tiny amount of sulfur which comes from the concentrated sulfuric acid. The C1s spectrum (Fig. 2b) can be fitted into a large peak of CeC/C]C at 284.5 eV and two small peaks of CeS/CeO at 286.3 eV and C]O at 288.9 eV [46], which reveals that carbons are mostly graphitic with a small fraction being oxidized. Two oxygen states of C]O at 531.8 eV and CeO at 533.0 eV are observed in the O1s spectrum (Fig. S1), indicating that CDs contain oxygenous groups [31]. The S2p spectrum (Fig. 2c) presents two sulfur components at 168.5 and 169.6 eV, being assigned to the 2p3/2 and 2p1/2 positions of sulfonic acid groups [47]. The interpretations of XPS are confirmed by the FTIR analysis. As shown in Fig. 2d, the distinctive band at 1617 cm−1 is ascribed to the aromatic C]C stretching vibrations. The bands at 3413, 1701, 1178, and 1036 cm−1 are attributed to the stretching vibrations of OeH, C] O, O]S]O, and SO3− groups [47]. The CeH stretching and bending vibrations are also seen at 2961, 2921, 1441, and 1399 cm−1. All the characterization data show the successful conversion of acetone into nanosized carbonaceous dots with aromatic clusters in the core and
The oxygen-containing organic solvents including acetone, ethanol, ethyl acetate, tetrahydrofuran, and diethyl ether were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Concentrated sulfuric acid (98%) was got from Nanjing Chemical Reagents Factory (Nanjing, China). Hemoglobin was purchased from Aladdin Chemical Co., Ltd. (Shanghai, China). All other reagents and chemicals were of analytical grade and used as received. Deionized water was used throughout the experiments. 2.2. Hot-injection synthesis of CDs CDs were fabricated by injecting various oxygen-containing organic solvents such as acetone, ethanol, ethyl acetate, tetrahydrofuran, and diethyl ether into hot concentrated sulfuric acid. In a typical synthesis, 2 mL of concentrated sulfuric acid was added into a 10-mL two-neck flask connected with a reflux condenser and heated to 170 °C. Then, 1 mL of acetone was quickly injected into the hot concentrated sulfuric acid, and the reaction mixture was kept at 170 °C for 1 min. With time, the color of the solution changed from colorless to yellow to dark brown, indicating the formation of CDs. After natural cooling, the mixture containing CDs was diluted with 50 mL of deionized water, followed by adjusting the pH to about 7 with NaOH. The CD dispersion was centrifuged at 6000 rpm for 10 min and finally purified by dialysis using a cellulose membrane (molecular weight cut off: 1000 Da) for 3 days. The synthetic procedures of CDs from ethanol, ethyl acetate, tetrahydrofuran, and diethyl ether were the same as those of acetone. 2.3. Fluorescence detection of Hb In a typical run, 70 mg of the acetone-derived CDs were dispersed in 1000 mL of phosphate buffer solution (200 mM, pH = 7). Then, 4500 μL of the Hb solution in a certain concentration was mixed with 500 μL of the above CD dispersion. The fluorescence spectrum with 340 nm excitation was recorded after the mixture was equilibrated for 30 s. Human blood samples, for determination of Hb concentrations, were pretreated prior to analysis according to the literature [43]. The 430
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Fig. 1. (a) TEM and high-resolution TEM (inset) images, (b) size distribution, (c) XRD pattern, and (d) Raman spectrum of CDs derived from acetone.
broad absorption in the UV–vis range with a shoulder at 273 nm, which is ascribed to the n–π* transition of the C]O bond [48]. The PL emission of CDs is critically dependent on the excitation wavelength. The strongest PL peak is centered at 401 nm on 340 nm excitation. The peak red-shifts from 391 to 559 nm when the excitation wavelength is tuned from 320 to 500 nm (Fig. 3a and b). Such an excitation-dependent emission behavior is common in fluorescent carbon materials,
oxygenous groups on the surface. 3.2. Optical properties of CDs As shown in the insets of Fig. 3a, the aqueous solution of CDs is pale yellow in daylight, and emits blue fluorescence under UV irradiation. The UV–vis absorption spectrum (Fig. 3a) exhibits that the CDs have a
Fig. 2. (a) XPS full scan, (b) C1s, (c) S2p, and FTIR spectra of CDs derived from acetone. 431
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Fig. 3. (a) UV–vis absorption and PL spectra of CDs derived from acetone. (b) The normalized PL spectra with excitation of 320–500 nm. Insets in (a): photographs of the CD aqueous solution excited by daylight and a UV lamp (365 nm).
Scheme 2. A possible formation mechanism of CDs from acetone through protonation, polymerization, and aromatization.
revealing that CDs have diverse emissive energy trap sites [17]. The PL quantum yield (QY) is estimated to be 5.67% against the reference of quinine sulfate, comparable to the previously reported values [11,25,49,50]. The PL intensity of CDs remains stable in high ionic strength medium (Fig. S2) and under continuous UV irradiation (Fig. S3), which is beneficial for their practical applications in complex biological systems.
3.3. Optimal synthetic conditions and a proposed formation mechanism of CDs We investigated the effects of reaction temperature and time on QY of CDs. It was found that at low temperature of 130 °C, acetone could not immediately convert into CDs because of the relatively weak dehydration and carbonization abilities of concentrated sulfuric acid; while at high temperature of 200 °C, black carbon foam generated due to the excessive carbonization of acetone. In the range of 140–180 °C, the maximum QY of CDs was obtained at 170 °C (Fig. S4). The reaction time was also important. A short time may bring about an imperfect inner core structure of CDs, whereas a long time probably damaged the surface groups, both of which decreased QY of CDs. As seen in Fig. S5, the optimal time for QY was 1 min. Based on experimental phenomenon and literature survey, we propose a possible formation mechanism of CDs (Scheme 2). In the presence of concentrated sulfuric acid, acetone accepts a proton, forming acetonium cation [51]. The acetonium cation is highly active, which can react with another acetone molecule to generate mesityl oxide [51]. In the following, the unsaturated mesityl oxide proceeds polymerization, producing oligomers with extended carbon chains [52]. Then, the oligomers curl and intertwine for further polycondensation and aromatization to create a number of carbogenic nanodots [52,53]. It should be noted that under the synthetic conditions, the reactions are rather complicated, and a variety of side products with unknown structures may produce. Thus, it is difficult to give the exact reaction paths of CDs from acetone.
Fig. 4. TEM images (left side), UV–vis absorption and PL spectra (right side) of CDs prepared from ethanol (a), ethyl acetate (b), tetrahydrofuran (c), and diethyl ether (d). Insets: photographs of the CD aqueous solution excited by daylight and a UV lamp (365 nm).
3.4. Various precursors for the synthesis of CDs using hot-injection strategy To check the general availability of the hot-injection strategy, besides acetone, we employed different oxygen-containing organic solvents including ethanol, ethyl acetate, tetrahydrofuran, and diethyl 432
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Table 1 Particle sizes, peaks in the XRD patterns, absorption peaks, emission wavelength, and QYs of CDs prepared from acetone, ethanol, ethyl acetate, tetrahydrofuran, and diethyl ether. Precursor
Particle sizes (nm)
XRD peaks (°)
Absorption peak (nm)
Emission wavelength (nm)
QY (%)
acetone ethanol ethyl acetate tetrahydrofuran diethyl ether
2–5 2–6 1–7 2–5 1–5
23.8, 22.4, 24.1, 22.5, 23.7,
273 267 265 275 263
391–559 447–554 457–550 409–553 449–551
5.67 3.85 5.26 2.19 3.67
42.7 44.9 42.9 41.9 42.9
Fig. 5. (a) UV–vis absorption spectrum of Hb and PL spectrum of the acetone-derived CDs. (b) The fluorescence decay profiles of CDs with and without of Hb (CHb = 360 nM). (c) PL spectra of CDs upon addition of different concentrations of Hb (from top to bottom: 0, 2, 10, 20, 50, 80, 120, 180, 240, 300, 360, 420, 500, 600, 700, and 800 nM). Inset in (c): The relationship between the fluorescence quenching ratio (I0eI)/I0 and Hb concentration. (d) Sensing selectivity of CDs toward Hb. All the concentrations of biomolecules are 360 nM. All the experiments are performed in phosphate buffer (20 mM, pH = 7). I and I0 refer to the fluorescence intensities of CDs at the peak position with and without biomolecule, respectively. CCDs = 7 mg L−1, λex = 340 nm.
because that these organic molecules can not accept the proton from concentrated sulfuric acid to form +OH group for the following dehydration process, which is indispensable during the synthetic procedures of CDs.
Table 2 The results of Hb detection in diluted human blood samples using the CD probe. The dilution factor (125,000-fold dilution) should be considered to obtain the final Hb concentrations in original blood samples. Diluted sample
Spiked (nM)
Found (nM)
Recovery (%)
RSD (%, n = 3)
male blood
0 20 50 100 0 20 50 100 0 20 50 100
14.7 32.7 62.8 119.2 18.6 38.3 64.3 125.2 17.6 40.3 68.9 111.7
– 94.2 97.1 103.9 – 99.2 93.7 105.6 – 107.2 101.9 95.0
3.7 3.3 2.9 4.5 2.6 3.5 4.3 3.2 4.1 2.8 3.5 5.1
female blood
child's blood
3.5. Detection of Hb The unique fluorescence property of CDs promotes us to explore their potential applications in biomolecule sensing. We note that the absorption band of Hb fully overlaps with the emission spectrum of CDs (Fig. 5a), implying that the inner filter effect (IFE) or the fluorescence resonance energy transfer (FRET) may occur between CDs and Hb [10]. As expected, the fluorescence of CDs can be quenched by Hb. To further investigate the quenching mechanism, the emission decay measurement is carried out, and it shows that the fluorescence lifetimes of CDs in the presence and absence of Hb are nearly identical (3.87 and 3.58 ns, respectively) (Fig. 5b), excluding the possibility of FRET and indicating the feasibility of IFE [54], especially due to the fact that Hb has a high molar extinction coefficient of 2.7 × 105 L mol−1 cm−1 at 406 nm which can result in a strong IFE efficiency. Under the optimal conditions (Figs. S8 and S9), the fluorescence intensity of CDs gradually decreases upon addition of increased concentration of Hb from 2 to 800 nM (Fig. 5c). The inset of Fig. 5c depicts the fluorescence change ratio (I0eI)/I0 as a function of Hb concentration. A nice linear relationship is observed in the region of 2–360 nM, and the regression equation is given as (I0eI)/I0 = 0.00182 × CHb (nM) + 0.00164 with a correlation coefficient R of 0.99. The limit of detection is calculated to be as low as 0.79 nM according to the rule of 3σ/slope. We evaluate the
ether as precursors to prepare CDs. All the small organic molecules could quickly convert into CDs with lateral sizes of 1–7 nm (Fig. 4, left panel). These CDs exhibit broad UV–vis absorption features and possess excitation-dependent emission properties with QYs of 2.19–5.26% (Fig. 4, right panel). They have disordered carbon structures and bear abundant oxygenous groups, which are confirmed by the XRD and FTIR results (Figs. S6 and S7). The detailed information about these CDs are listed in Table 1. It is worth mentioning that the organic solvents without an oxygen atom such as benzene, chloroform, and acetonitrile, are unsuitable precursors towards fast fabrication of CDs, probably
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interference effects of several representative biomolecules, such as glutathione (GSH), cysteine (Cys), uric acid (UA), ascorbic acid (AA), dopamine (DA), urea, glucose, lysozyme, and transferrin, and find that these compounds cause negligible fluorescence quenching under the same conditions as used for Hb detection (Fig. 5d), indicating the excellent sensing selectivity of CDs toward Hb. Compared with the previous approaches for Hb detection (Table S1) [36,38–40,42,55], the CD based fluorescence method shows outstanding performance in terms of high sensitivity, good selectivity, easy material preparation, and low cost. For real applications, the CD probe is employed to quantify the Hb concentration in human blood samples following the protocol described in the experimental section. The Hb concentrations in three blood samples of healthy male, female adults and a child are determined to be 1.84 ± 0.07, 2.33 ± 0.06, and 2.20 ± 0.09 mM, respectively, agreeing well with the normal Hb levels [55]. Using the standard addition method, the recovery of 93.7–107.2% and the relative standard deviation (RSD) of 2.6–5.1% are recorded in Table 2, indicating the proposed method for Hb sensing in complex biological systems is reliable. For comparison, the commercial immunoturbidimetric assay is used to estimate the amount of Hb in the same blood samples. As shown in Table S2, the Hb concentrations determined by two different methods are quite close, further revealing that the CD probe holds great potential for Hb analysis.
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4. Conclusions In conclusion, we develop a facile hot-injection strategy for 1-min conversion of various oxygen-containing organic solvents including acetone, ethanol, ethyl acetate, tetrahydrofuran, and diethyl ether into CDs with the assistance of concentrated sulfuric acid. The resultant CDs have disordered graphite structures, bear abundant oxygenous groups, and possess unique optical properties, which can serve as a promising fluorescence probe for highly sensitive and selective detection of Hb with a linear range of 2–360 nM and a limit of detection as low as 0.79 nM. After careful investigations, the inner filter effect is proposed to explain the sensing mechanism. The utilization of CDs for Hb assay in human blood samples achieves good results, showing their great applicability. Our work provides an alternative route toward rapid and low-cost synthesis of CDs from the most common precursors without need of special equipment, and extends the biomolecule sensing applications of CDs. Acknowledgements The authors gratefully acknowledge the financial support from the Natural Science Foundation of Ningbo City (2017A610230), the K. C. Wong Magna Fund in Ningbo University, and the China Postdoctoral Science Foundation (2016M592026). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dyepig.2019.03.001. References [1] Hola K, Zhang Y, Wang Y, Giannelis EP, Zboril R, Rogach AL. Carbon dots—emerging light emitters for bioimaging, cancer therapy and optoelectronics. Nano Today 2014;9:590–603. [2] Yuan FL, Li SH, Fan ZT, Meng XY, Fan LZ, Yang SH. Shining carbon dots: synthesis and biomedical and optoelectronic applications. Nano Today 2016;11(5):565–86. [3] Baker SN, Baker GA. Luminescent carbon nanodots: emergent nanolights. Angew Chem Int Ed 2010;49:6726–44. [4] Li HT, Kang ZH, Liu Y, Lee ST. Carbon nanodots: synthesis, properties and applications. J Mater Chem 2012;22:24230–53. [5] Zhao AD, Chen ZW, Zhao CQ, Gao N, Ren JS, Qu XG. Recent advances in bioapplications of C-dots. Carbon 2015;85:309–27.
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Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
Hot-injection strategy for 1-min synthesis of carbon dots from oxygencontaining organic solvents: Toward fluorescence sensing of hemoglobin
T
Yaoping Hua,∗, Zhijin Gaob a b
School of Materials Science and Chemical Engineering, Ningbo University, Ningbo, 315211, China Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo, 315201, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Hot-injection One-minute synthesis Carbon dots Oxygen-containing organic solvents Sensing of hemoglobin
Here, we present a rapid and general fabrication methodology for fluorescent carbon dots (CDs) via injecting various oxygen-containing organic solvents including acetone, ethanol, ethyl acetate, tetrahydrofuran, and diethyl ether into hot concentrated sulfuric acid. All these small organic molecules are recognized as common, cheap, and abundant carbon precursors, which can convert into CDs just in 1 min through protonation, polymerization and aromatization. The as-prepared CDs with disordered amorphous cores and plentiful oxygenous groups show fascinating photoluminescence features, that can be used for sensitive and selective detection of hemoglobin (Hb) based on fluorescence quenching. The linear detection range is estimated to be 2–360 nM and the limit of detection is as low as 0.79 nM. After careful investigations, the inner filter effect is proposed to explain the sensing mechanism because the absorption band of Hb fully covers the emission spectrum of CDs and the fluorescence lifetime of CDs remains nearly constant after addition of Hb. The sensor is successfully applied for Hb detection in human blood samples, exhibiting a great potential for quantitative analysis of Hb in complex biological matrices.
1. Introduction Carbon dots (CDs) represent a new family of fluorescent carbogenic materials, generally containing small aromatic carbon clusters in the core and being functionalized with abundant oxygenous groups at the surface [1,2]. Taking advantages of alluring optical properties, low toxicity, robust chemical inertness, and facile modification [3–6], CDs provide unprecedented opportunities for applications in bioimaging, sensors, nanomedicine, photocatalysis, optoelectronics, etc [7–15]. Since their accident discovery as byproducts during electrophoretic purification of single-walled carbon nanotubes [16], numerous approaches including laser ablation [17], electrochemical exfoliation [18–20], acidic oxidation [21,22], thermal oxidation [23,24], ultrasonic treatment [25,26], microwave heating [27–30], and hydrothermal technique [31–35], have been explored to fabricate CDs using a broad range of carbon feedstocks. However, except microwave method, other above-mentioned approaches usually suffer from a long reaction time that lasts up to several hours or even a few days. In addition, some synthetic approaches require special instruments, or uncommon reagents, or complicated procedures, which restrict their widespread usage. Currently, despite many impressive advances, the rapid synthesis of CDs from cheap carbon sources with simple apparatus is still actively
∗
pursued. Hemoglobin (Hb), containing heme groups, is a metalloprotein in the red blood cells of vertebrates. It transports oxygen from the lungs to the rest of the body to permit aerobic respiration. The deficiency of Hb decreases blood oxygen-carrying capacity, causing symptoms of anemia, while the high levels of Hb may result in lung disease and certain tumors [36,37]. Therefore, the accurate determination of the trace amount of Hb in biosystems is of great significance for medical diagnosis to control the related clinical illness. Unfortunately, the recently reported methods for Hb detection like colorimetric approaches [38], spectrophotometric assays [36,39,40], electrochemical measurements [37,41], and chemiluminescence determination [42] have some drawbacks such as the requirement of expensive materials, the need of tedious sample pretreatment, and relatively high detection limit. At present, it is highly demanded to develop feasible methods for simple, cost-effective, and sensitive detection of Hb in complex biological media. Herein, we report a novel strategy for rapid synthesis of fluorescent CDs by injecting various oxygen-containing organic solvents into hot concentrated sulfuric acid (Scheme 1). The selected solvents including acetone, ethanol, ethyl acetate, tetrahydrofuran, and diethyl ether, serve as common, cheap, and abundant precursors, all of which can
Corresponding author. E-mail address: [email protected] (Y. Hu).
https://doi.org/10.1016/j.dyepig.2019.03.001 Received 1 February 2019; Received in revised form 26 February 2019; Accepted 1 March 2019 Available online 05 March 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
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Scheme 1. A diagram about the rapid synthesis of CDs from oxygen-containing organic solvents using hot-injection strategy, and the application of CDs for Hb sensing.
whole blood samples were first diluted 100,000-fold in deionized water, and then sonicated for 30 s to release Hb from erythrocytes. After centrifugation at 3000 rpm for 10 min, the as-prepared blood sample (4000 μL) was mixed with the standard Hb solution in a certain concentration (500 μL) and the CD dispersion (500 μL). The fluorescence measurement was performed as described above. All the tests were conducted in triplicate.
efficiently convert into CDs in 1 min without need of any special equipment. The resultant acetone-derived CDs with partially disordered carbon structures exhibit a highly selective and sensitive detection of Hb through a fluorescence quenching process. After detailed investigations, the inner filter effect is proposed to elucidate the “on-off” sensing mechanism. Moreover, the fluorescence sensor has been successfully applied for the assay of Hb in human blood samples, and satisfactory results are achieved.
3. Results and discussion
2. Experimental
3.1. Morphology, structure and composition of CDs
2.1. Chemicals
As a sole carbon source, acetone (1 mL) was rapidly injected into the hot concentrated sulfuric acid (2 mL) at 170 °C, and the reaction at that temperature lasted for only 1 min, immediately producing a dark brown dispersion of CDs. The TEM image (Fig. 1a) shows that the as-synthesized CDs are nearly spherical and well separated from each other. They have a narrow size distribution ranging from 2.0 to 5.0 nm with the maximum population of approximately 3.5 nm (Fig. 1b). The high-resolution TEM image (Fig. 1a, inset) reveals the amorphous nature of CDs without clear lattice fringes. The XRD pattern (Fig. 1c) displays two broad diffraction peaks at 2θ angles of about 23.8° and 42.7°, which are attributable to amorphous carbon composed of aromatic carbon clusters oriented in a considerably random fashion [44]. In the Raman spectrum (Fig. 1d), the D band at 1358 cm−1 stands for the sp3 carbon species, and the G band at 1579 cm−1 represents the sp2 carbon networks. The coexistence of D and G bands with intensity ratio of 0.71 reflects some disorder or defects in the carbon matrix [45]. The elemental compositions and chemical bonds of CDs were investigated by the XPS measurement. The survey spectrum (Fig. 2a) shows sharp C1s, O1s and weak S2s, S2p signals. The atomic ratios of C, O, and S are 67.4%, 29.0%, and 3.6% respectively, demonstrating that the sample comprises mainly carbon and oxygen, as well as a tiny amount of sulfur which comes from the concentrated sulfuric acid. The C1s spectrum (Fig. 2b) can be fitted into a large peak of CeC/C]C at 284.5 eV and two small peaks of CeS/CeO at 286.3 eV and C]O at 288.9 eV [46], which reveals that carbons are mostly graphitic with a small fraction being oxidized. Two oxygen states of C]O at 531.8 eV and CeO at 533.0 eV are observed in the O1s spectrum (Fig. S1), indicating that CDs contain oxygenous groups [31]. The S2p spectrum (Fig. 2c) presents two sulfur components at 168.5 and 169.6 eV, being assigned to the 2p3/2 and 2p1/2 positions of sulfonic acid groups [47]. The interpretations of XPS are confirmed by the FTIR analysis. As shown in Fig. 2d, the distinctive band at 1617 cm−1 is ascribed to the aromatic C]C stretching vibrations. The bands at 3413, 1701, 1178, and 1036 cm−1 are attributed to the stretching vibrations of OeH, C] O, O]S]O, and SO3− groups [47]. The CeH stretching and bending vibrations are also seen at 2961, 2921, 1441, and 1399 cm−1. All the characterization data show the successful conversion of acetone into nanosized carbonaceous dots with aromatic clusters in the core and
The oxygen-containing organic solvents including acetone, ethanol, ethyl acetate, tetrahydrofuran, and diethyl ether were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Concentrated sulfuric acid (98%) was got from Nanjing Chemical Reagents Factory (Nanjing, China). Hemoglobin was purchased from Aladdin Chemical Co., Ltd. (Shanghai, China). All other reagents and chemicals were of analytical grade and used as received. Deionized water was used throughout the experiments. 2.2. Hot-injection synthesis of CDs CDs were fabricated by injecting various oxygen-containing organic solvents such as acetone, ethanol, ethyl acetate, tetrahydrofuran, and diethyl ether into hot concentrated sulfuric acid. In a typical synthesis, 2 mL of concentrated sulfuric acid was added into a 10-mL two-neck flask connected with a reflux condenser and heated to 170 °C. Then, 1 mL of acetone was quickly injected into the hot concentrated sulfuric acid, and the reaction mixture was kept at 170 °C for 1 min. With time, the color of the solution changed from colorless to yellow to dark brown, indicating the formation of CDs. After natural cooling, the mixture containing CDs was diluted with 50 mL of deionized water, followed by adjusting the pH to about 7 with NaOH. The CD dispersion was centrifuged at 6000 rpm for 10 min and finally purified by dialysis using a cellulose membrane (molecular weight cut off: 1000 Da) for 3 days. The synthetic procedures of CDs from ethanol, ethyl acetate, tetrahydrofuran, and diethyl ether were the same as those of acetone. 2.3. Fluorescence detection of Hb In a typical run, 70 mg of the acetone-derived CDs were dispersed in 1000 mL of phosphate buffer solution (200 mM, pH = 7). Then, 4500 μL of the Hb solution in a certain concentration was mixed with 500 μL of the above CD dispersion. The fluorescence spectrum with 340 nm excitation was recorded after the mixture was equilibrated for 30 s. Human blood samples, for determination of Hb concentrations, were pretreated prior to analysis according to the literature [43]. The 430
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Fig. 1. (a) TEM and high-resolution TEM (inset) images, (b) size distribution, (c) XRD pattern, and (d) Raman spectrum of CDs derived from acetone.
broad absorption in the UV–vis range with a shoulder at 273 nm, which is ascribed to the n–π* transition of the C]O bond [48]. The PL emission of CDs is critically dependent on the excitation wavelength. The strongest PL peak is centered at 401 nm on 340 nm excitation. The peak red-shifts from 391 to 559 nm when the excitation wavelength is tuned from 320 to 500 nm (Fig. 3a and b). Such an excitation-dependent emission behavior is common in fluorescent carbon materials,
oxygenous groups on the surface. 3.2. Optical properties of CDs As shown in the insets of Fig. 3a, the aqueous solution of CDs is pale yellow in daylight, and emits blue fluorescence under UV irradiation. The UV–vis absorption spectrum (Fig. 3a) exhibits that the CDs have a
Fig. 2. (a) XPS full scan, (b) C1s, (c) S2p, and FTIR spectra of CDs derived from acetone. 431
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Fig. 3. (a) UV–vis absorption and PL spectra of CDs derived from acetone. (b) The normalized PL spectra with excitation of 320–500 nm. Insets in (a): photographs of the CD aqueous solution excited by daylight and a UV lamp (365 nm).
Scheme 2. A possible formation mechanism of CDs from acetone through protonation, polymerization, and aromatization.
revealing that CDs have diverse emissive energy trap sites [17]. The PL quantum yield (QY) is estimated to be 5.67% against the reference of quinine sulfate, comparable to the previously reported values [11,25,49,50]. The PL intensity of CDs remains stable in high ionic strength medium (Fig. S2) and under continuous UV irradiation (Fig. S3), which is beneficial for their practical applications in complex biological systems.
3.3. Optimal synthetic conditions and a proposed formation mechanism of CDs We investigated the effects of reaction temperature and time on QY of CDs. It was found that at low temperature of 130 °C, acetone could not immediately convert into CDs because of the relatively weak dehydration and carbonization abilities of concentrated sulfuric acid; while at high temperature of 200 °C, black carbon foam generated due to the excessive carbonization of acetone. In the range of 140–180 °C, the maximum QY of CDs was obtained at 170 °C (Fig. S4). The reaction time was also important. A short time may bring about an imperfect inner core structure of CDs, whereas a long time probably damaged the surface groups, both of which decreased QY of CDs. As seen in Fig. S5, the optimal time for QY was 1 min. Based on experimental phenomenon and literature survey, we propose a possible formation mechanism of CDs (Scheme 2). In the presence of concentrated sulfuric acid, acetone accepts a proton, forming acetonium cation [51]. The acetonium cation is highly active, which can react with another acetone molecule to generate mesityl oxide [51]. In the following, the unsaturated mesityl oxide proceeds polymerization, producing oligomers with extended carbon chains [52]. Then, the oligomers curl and intertwine for further polycondensation and aromatization to create a number of carbogenic nanodots [52,53]. It should be noted that under the synthetic conditions, the reactions are rather complicated, and a variety of side products with unknown structures may produce. Thus, it is difficult to give the exact reaction paths of CDs from acetone.
Fig. 4. TEM images (left side), UV–vis absorption and PL spectra (right side) of CDs prepared from ethanol (a), ethyl acetate (b), tetrahydrofuran (c), and diethyl ether (d). Insets: photographs of the CD aqueous solution excited by daylight and a UV lamp (365 nm).
3.4. Various precursors for the synthesis of CDs using hot-injection strategy To check the general availability of the hot-injection strategy, besides acetone, we employed different oxygen-containing organic solvents including ethanol, ethyl acetate, tetrahydrofuran, and diethyl 432
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Table 1 Particle sizes, peaks in the XRD patterns, absorption peaks, emission wavelength, and QYs of CDs prepared from acetone, ethanol, ethyl acetate, tetrahydrofuran, and diethyl ether. Precursor
Particle sizes (nm)
XRD peaks (°)
Absorption peak (nm)
Emission wavelength (nm)
QY (%)
acetone ethanol ethyl acetate tetrahydrofuran diethyl ether
2–5 2–6 1–7 2–5 1–5
23.8, 22.4, 24.1, 22.5, 23.7,
273 267 265 275 263
391–559 447–554 457–550 409–553 449–551
5.67 3.85 5.26 2.19 3.67
42.7 44.9 42.9 41.9 42.9
Fig. 5. (a) UV–vis absorption spectrum of Hb and PL spectrum of the acetone-derived CDs. (b) The fluorescence decay profiles of CDs with and without of Hb (CHb = 360 nM). (c) PL spectra of CDs upon addition of different concentrations of Hb (from top to bottom: 0, 2, 10, 20, 50, 80, 120, 180, 240, 300, 360, 420, 500, 600, 700, and 800 nM). Inset in (c): The relationship between the fluorescence quenching ratio (I0eI)/I0 and Hb concentration. (d) Sensing selectivity of CDs toward Hb. All the concentrations of biomolecules are 360 nM. All the experiments are performed in phosphate buffer (20 mM, pH = 7). I and I0 refer to the fluorescence intensities of CDs at the peak position with and without biomolecule, respectively. CCDs = 7 mg L−1, λex = 340 nm.
because that these organic molecules can not accept the proton from concentrated sulfuric acid to form +OH group for the following dehydration process, which is indispensable during the synthetic procedures of CDs.
Table 2 The results of Hb detection in diluted human blood samples using the CD probe. The dilution factor (125,000-fold dilution) should be considered to obtain the final Hb concentrations in original blood samples. Diluted sample
Spiked (nM)
Found (nM)
Recovery (%)
RSD (%, n = 3)
male blood
0 20 50 100 0 20 50 100 0 20 50 100
14.7 32.7 62.8 119.2 18.6 38.3 64.3 125.2 17.6 40.3 68.9 111.7
– 94.2 97.1 103.9 – 99.2 93.7 105.6 – 107.2 101.9 95.0
3.7 3.3 2.9 4.5 2.6 3.5 4.3 3.2 4.1 2.8 3.5 5.1
female blood
child's blood
3.5. Detection of Hb The unique fluorescence property of CDs promotes us to explore their potential applications in biomolecule sensing. We note that the absorption band of Hb fully overlaps with the emission spectrum of CDs (Fig. 5a), implying that the inner filter effect (IFE) or the fluorescence resonance energy transfer (FRET) may occur between CDs and Hb [10]. As expected, the fluorescence of CDs can be quenched by Hb. To further investigate the quenching mechanism, the emission decay measurement is carried out, and it shows that the fluorescence lifetimes of CDs in the presence and absence of Hb are nearly identical (3.87 and 3.58 ns, respectively) (Fig. 5b), excluding the possibility of FRET and indicating the feasibility of IFE [54], especially due to the fact that Hb has a high molar extinction coefficient of 2.7 × 105 L mol−1 cm−1 at 406 nm which can result in a strong IFE efficiency. Under the optimal conditions (Figs. S8 and S9), the fluorescence intensity of CDs gradually decreases upon addition of increased concentration of Hb from 2 to 800 nM (Fig. 5c). The inset of Fig. 5c depicts the fluorescence change ratio (I0eI)/I0 as a function of Hb concentration. A nice linear relationship is observed in the region of 2–360 nM, and the regression equation is given as (I0eI)/I0 = 0.00182 × CHb (nM) + 0.00164 with a correlation coefficient R of 0.99. The limit of detection is calculated to be as low as 0.79 nM according to the rule of 3σ/slope. We evaluate the
ether as precursors to prepare CDs. All the small organic molecules could quickly convert into CDs with lateral sizes of 1–7 nm (Fig. 4, left panel). These CDs exhibit broad UV–vis absorption features and possess excitation-dependent emission properties with QYs of 2.19–5.26% (Fig. 4, right panel). They have disordered carbon structures and bear abundant oxygenous groups, which are confirmed by the XRD and FTIR results (Figs. S6 and S7). The detailed information about these CDs are listed in Table 1. It is worth mentioning that the organic solvents without an oxygen atom such as benzene, chloroform, and acetonitrile, are unsuitable precursors towards fast fabrication of CDs, probably
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interference effects of several representative biomolecules, such as glutathione (GSH), cysteine (Cys), uric acid (UA), ascorbic acid (AA), dopamine (DA), urea, glucose, lysozyme, and transferrin, and find that these compounds cause negligible fluorescence quenching under the same conditions as used for Hb detection (Fig. 5d), indicating the excellent sensing selectivity of CDs toward Hb. Compared with the previous approaches for Hb detection (Table S1) [36,38–40,42,55], the CD based fluorescence method shows outstanding performance in terms of high sensitivity, good selectivity, easy material preparation, and low cost. For real applications, the CD probe is employed to quantify the Hb concentration in human blood samples following the protocol described in the experimental section. The Hb concentrations in three blood samples of healthy male, female adults and a child are determined to be 1.84 ± 0.07, 2.33 ± 0.06, and 2.20 ± 0.09 mM, respectively, agreeing well with the normal Hb levels [55]. Using the standard addition method, the recovery of 93.7–107.2% and the relative standard deviation (RSD) of 2.6–5.1% are recorded in Table 2, indicating the proposed method for Hb sensing in complex biological systems is reliable. For comparison, the commercial immunoturbidimetric assay is used to estimate the amount of Hb in the same blood samples. As shown in Table S2, the Hb concentrations determined by two different methods are quite close, further revealing that the CD probe holds great potential for Hb analysis.
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