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In situ photochemical synthesis of fluorescent carbon dots for optical sensing of hydrogen peroxide and antioxidants
Author’s Accepted Manuscript In situ photochemical synthesis of fluorescent carbon dots for optical sensing of hydrogen peroxide and antioxidants Isabel Costas-Mora, Vanesa Romero, Isela Lavilla, Carlos Bendicho www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(15)30220-4 http://dx.doi.org/10.1016/j.talanta.2015.07.093 TAL15858
To appear in: Talanta Received date: 10 June 2015 Revised date: 27 July 2015 Accepted date: 31 July 2015 Cite this article as: Isabel Costas-Mora, Vanesa Romero, Isela Lavilla and Carlos Bendicho, In situ photochemical synthesis of fluorescent carbon dots for optical sensing of hydrogen peroxide and antioxidants, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.07.093 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.
In situ photochemical synthesis of fluorescent carbon dots for optical sensing of hydrogen peroxide and antioxidants Isabel Costas-Mora, Vanesa Romero, Isela Lavilla and Carlos Bendicho* Departamento de Química Analítica y Alimentaria, Área de Química Analítica, Facultad de Química, Universidad de Vigo, Campus As Lagoas-Marcosende s/n, 36310 Vigo, Spain. *Corresponding author. Tel.: +34-986-812281; fax: +34-986-812556; e-mail: [email protected] Abstract A new synthesis approach for obtaining fluorescent carbon dots (CDs) based on UV irradiation of carbohydrates was developed. The photochemical synthesis pathway allows the formation of water soluble CDs of analytical usefulness within one min. CDs obtained by photochemical treatment from the sucrose/NaOH/poly(ethylene glycol) system are monodisperse with an average size of 8 nm as determined by transmission electron microscopy. A dramatic increase in the CDs fluorescence (turn on) is observed when H2O2 is added. The decrease in CDs size occurring by the action of highly oxidant OH radicals gives rise to confinement of emissive energy traps and, in turn, to fluorescence enhancement. Antioxidants such as ascorbic acid and glutathione inhibit the photochemical reaction giving rise to a decrease in fluorescence of the CDs/H2O2 system (turn on-off). The detection limit was 5 µM H2O2 and the repeatability expressed as the relative standard deviation was 3.8% (N=7). The photochemical synthesis of CDs allows building a green, low-cost, safe and fast assay for the detection of H2O2 and 1
antioxidants. An application of the novel fluorescent nanoprobe to H2O2 detection in contact lens cleaning solutions is performed. Keywords: carbon dots; fluorescence; nanoprobe; photochemical synthesis; hydrogen peroxide; antioxidants.
1. Introduction Fluorescent nanoparticles (NPs) have been increasingly used as optical probes for the detection of a wide variety of chemical species [1-3]. In the last years, carbon dots (CDs) have emerged as one of the most interesting optical nanoprobes due to their high aqueous stability, easy functionalization and low toxicity [4]. CDs are fluorescent nanoparticles with sizes comprised between 2-10 nm, which can be synthesized from natural and non-toxic precursors such as carbohydrates. So far, synthesis pathways for obtaining fluorescent CDs include laser ablation, arcdischarge, electrochemical exfoliation, microwave treatment, thermal carbonization, acid dehydration, etc. [4, 5]. However, most synthesis methods have some drawbacks such as long operation time and harsh synthesis conditions, i.e. high temperature, so new approaches should be investigated in order to develop greener synthesis methods. Some studies have been focused on the use of greener carbon sources as CDs precursors such as pomelo, watermelon and banana peels [6-8], pepper [9] or bamboo leaves [10], which cause a low environmental impact and are cost-effective, yet the reaction time and temperature used to obtain fluorescent CDs remain a drawback. Generally, fluorescent NPs-based assays described in the literature for sensing different chemical species require two-step, i.e., firstly fluorescent NPs are synthesized following 2
different procedures, and secondly, a recognition event is needed for detection of the target analyte. Recently, our research group has developed a new detection strategy for methylmercury based on the integration of sonochemical synthesis of CDs and sensing within a single step [11]. This approach eliminates the need for large and tedious procedures for purification and stabilization of freshly synthesized CDs, and moreover, the time required to accomplish the assay is remarkably shortened. H2O2 is a highly reactive oxidant often used in water treatment plants as well as in household products due to its antimicrobial properties. Its use is especially relevant in contact lens cleaning solutions in order to eliminate pathogens by oxidative process. Nevertheless, H2O2 levels need to be controlled in those samples, since H2O2 can be harmful to the ocular epithelium and cornea causing eyes irritation and possible corneal damage [12]. To date, most of the H2O2 detection methods are based on the use of enzymes, which pose some limitations such as high cost and stability problems due to changes in temperature, pH and the presence of concomitants. In recent years, an increasing number of nanostructured materials have been implemented in nonenzymatic assays for H2O2 detection. Thus, nanocomposites [13, 14], metallic nanoparticles [15], inorganic nanoparticles [16] and QDs [17], have been used for monitoring H2O2 in household products and contact lens cleaning solutions. Although the CDs ability to detect H2O2 has already been reported [18-22], those methods were mainly applied to detect H2O2 in biological matrices such as live cells, zebrafish larvae, serum and saliva [20-22]. So far, no application has appeared for the determination of H2O2 in household products and contact lens cleaning solutions. In this work, synthesis of fluorescent CDs upon application of UV radiation to carbohydrates is demonstrated for the first time. Enhancement/quenching effects of the
3
CDs fluorescence caused by the presence of H2O2 and antioxidants (e.g., ascorbic acid and glutathione) in the reaction medium can be the basis for a fast, low cost and sensitive fluorescent assay, which integrates synthesis and sensing in one step. Application of the novel assay for the detection of H2O2 in contact lens cleaning solutions is provided. 2. Experimental Section 2.1. Reagents and Chemicals Ultrapure water obtained from an Ultra Clear TWF EDI UV TM system (Siemens AG, Barsbuettel, Germany) was used for the preparation of working solutions. D-glucose, D-fructose, sucrose, (Sigma-Aldrich, St. Louis, MO, USA) and starch (Probus, Badalona, Barcelona) were tested as carbon sources. Several species purchased from Sigma-Aldrich were tried as stabilizing agents including poly(ethylene glycol) (PEG, MW=200), Triton X-100,Triton X-114, Tween 80, polyethylenimine (PEI), hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), polyvinylpyrrolidone (PVP), bovine serum albumin (BSA), Lcysteine, glutathione (GSH) and citric acid. NaOH was obtained from Prolabo (Paris, France). Potassium permanganate (Carlo Erba, Italy) and sulfuric acid (Prolabo) were used in order to validate the reliability of the fluorescent assay. The following compounds were tested as potential interferents: NaCl, sodium tetraborate,
sodium
citrate,
calcium
4
carbonate
(Sigma-Aldrich),
ethylenediaminetetraacetic acid (EDTA) (Prolabo), sodium phosphate (Panreac, Barcelona, Spain). 2.2. Apparatus Photochemical treatments were carried out in open quartz tubes (15 mm i.d., 125 mm height) using a 705 UV digester (Metrohm) equipped with a high-pressure mercury lamp (500 W). In addition, it is equipped with a holder for twelve tubes. A Thermo Scientific NanoDrop 3300 Fluorospectrometer whose technical specifications and operation mode have been outlined in earlier works [23, 24] was used to carry out fluorescence measurements. The fluorescence intensity at 517 nm was measured after excitation at a wavelength of 470 nm using blue LED. It should be mentioned that this miniaturized instrument can operate through connection to a computer via USB, thus facilitating field analysis. The effect of the excitation wavelength over the emission wavelength was studied using a Shimadzu RF-1501 Spectrofluorometer. In order to characterize CDs synthesized by photochemical treatment, UV-Vis absorption
measurements
were
performed
using
a
NanoDrop
ND-1000
spectrophotometer whose operation mode was described in an earlier work [25]. Transmission electron microscopy (TEM) was performed with JEOL JEM-1010 microscope operating at an acceleration voltage of 100 kV. TEM experiments were made by dropping the sample onto a carbon-coated copper grid. Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet 6700 spectrometer within the range of 400-4000 cm−1 with a resolution of 4 cm−1.
5
2.3 Experimental procedure for building the fluorescent nanoprobe Sucrose (20 mg) was introduced in a quartz tube and then, 1.5 mL of aqueous sample containing H2O2 was added. Once sucrose was solubilized, 0.8 mL of PEG and 200 µL of NaOH 3 M were added to the mixture. After that, the tubes were introduced in the sample holder of the UV digester and irradiated for 1 min. The color of the bulk solution changed from clear to light yellow. Finally, a 2 μL aliquot of the sample was placed onto the pedestal of the microfluorospectrometer in order to measure the fluorescence signal. A blank experiment was performed before each measurement in order to calculate analytical response (I/I0, where I0 and I represents the fluorescence intensity of CDs synthesized in the absence and in the presence of H2O2, respectively).
3. Results and discussion 3.1. Optimization of experimental parameters influencing the fluorescent assay In order to assess the effects of the different variables involved in the proposed system for H2O2 detection, the 2 factorial fractional design was applied for screening optimization [26]. This design allows assessing the effect of 4 variables (i.e. sucrose mass, PEG concentration, NaOH concentration and UV irradiation time) by performing 8 experiments in duplicate. The significance of the studied variables was established by comparison with the experimental error, calculated as two times the average standard deviation of all experiments (2 s̅ ). Standardized effects showed in Fig. S-1 reveal that all studied variables had a significant effect, so the assay was optimized in more detail following the univariate approach. 6
3.1.1. Selection of the carbon source and its concentration Carbohydrates have proved to be suitable precursors for building CDs following different synthetic routes. In studies concerning the removal of carbohydrates by UV irradiation, it has been shown that photodegradation occurs in alkaline medium [27]. In this work, different carbohydrates were tested as CDs precursors including D-glucose, D-fructose, sucrose and starch. It was observed that CDs can be synthesized from different carbon precursors using different irradiation times. The results obtained with 30 mg of each carbohydrate are shown in Fig. 1. As can be observed (intrinsic fluorescence signal, dotted line), fluorescent CDs can be synthesized using any of the tested carbohydrates, but different reaction kinetics were observed with each carbon precursor. Therefore, the use of simple sugars such as glucose and fructose allows synthesizing CDs in very short irradiation times, i.e. 45 and 30 s, respectively. The use of sucrose or starch requires longer irradiation times (i.e. 60 s) to achieve fluorescent CDs. The behavior of starch can be ascribed to the large number of glucose units of this polysaccharide. Sucrose is a disaccharide composed of glucose and fructose units, so higher energy is required to break down its structure, and hence, a longer irradiation time is required. Given that all carbohydrates tried lead to a rapid synthesis of fluorescent CDs, all of them were tested as fluorescent nanoprobes for H2O2 detection. As is shown in Fig. 1 (analytical response, solid line), the I/I0 ratio depends on both the carbon source and irradiation time. In all cases, a remarkable increase in CDs fluorescence is observed in the presence of H2O2. For glucose, fructose and starch, a maximum analytical response close to I/I0~1.5 is obtained in a short time (i.e., 30-90 s). Nevertheless, the use of sucrose as CDs precursor allows achieving a significant improvement of the analytical 7
response (I/I0~2) using an irradiation time of 2 min. In view of the obtained results, sucrose was selected as carbon source and its concentration was optimized in order to reach an adequate sensitivity for H2O2 detection. The effect of sucrose mass was studied in the range of 5-60 mg. Fig. 2(A) shows both the intrinsic fluorescence intensity of CDs and analytical responses for H2O2 detection. As can be observed, the best analytical response is obtained using a sucrose mass in the range of 20-25 mg. 3.1.2. Selection of the stabilizing agent and its concentration Stabilizing agents may influence the optical properties of CDs [28, 29]. For this reason, several stabilizing agents including PEG, triton X-100, triton X-114, tween 80, PEI, CTAB, SDS, PVP, BSA, L-cysteine, GSH and citric acid were tested to obtain suitable fluorescent CDs for the detection of H2O2. As is shown in Fig. 2(B), the addition of PEG, tween 80 or BSA prior to UV irradiation allows obtaining CDs with increased fluorescence as compared to CDs synthesized in the absence of stabilizing agent. For this reason, different concentrations of stabilizing agents (PEG, tween 80 and BSA) were tried for the detection of H2O2. As is shown in Fig. 2(C), an optimal analytical response for H2O2 was only obtained when PEG was added to the reaction medium. An increase in the PEG concentration up to 30 % (v/v) led to an increased analytical signal. Therefore, a PEG concentration of 35 % (v/v) was chosen for further experiments. 3.1.3. Effect of NaOH concentration There are several studies pointing out that pH is a critical parameter in order to get fluorescent CDs [10, 11, 30]. In this work, it was observed that alkaline medium is mandatory in order to obtain fluorescent CDs, since photodegradation of carbohydrates requires an alkaline medium [27]. For this reason, the effect of NaOH concentration
8
added was studied in the range of 1–6 M (Fig. 3A). A NaOH concentration of 3 M was chosen in order to reach the best analytical response. 3.1.4. Effect of UV irradiation time Irradiation times ranging from 45 to 210 s were applied. As is shown in Fig. 3(B), an increased analytical response for H2O2 is obtained using a short irradiation time. An irradiation time longer than 120 s causes the analytical signal to decrease. In view of the obtained results, a time of one min was selected as optimum irradiation time. These conditions allow not only the sensitive detection of H2O2 but also a high sample throughput, since 12 samples can be run per min. 3.2. Study of potential interferences In order to investigate the effect of potential matrix interferences in the sample, the fluorescence enhancement caused by 1 mM of H2O2 in the presence of several foreign substances was assessed. An interference effect was considered significant when the analytical response varied beyond ±10%. Several compounds commonly present in contact lens cleaning solutions were evaluated as potential interferences. Analytical results for interference studies are shown in Table S-1. Depressive effects on the analytical response were observed for sodium citrate, sodium tetraborate, EDTA and sodium phosphate at a concentration higher than 25, 3, 0.1 and 3.5% (m/v), respectively. On the other hand, it was found that sodium chloride caused a positive effect at a concentration higher than 7% (m/v). Carbonate was also tested as a potential interference because it is a potent OH radical scavenger. It was observed that carbonate concentrations higher than 0.3 M caused a decreased analytical
9
signal. It must be highlighted that the tolerance limits found in the interference study are higher than the concentrations usually present in contact lens cleaning solutions. 3.3. Analytical characteristics of the fluorescent nanoprobe Analytical figures of merit of the fluorescent assay were established under optimal conditions. When CDs synthesis is performed in the presence of H2O2, a significant fluorescence enhancement occurs and no spectral shift of the emission band is observed. A linear relationship between I/I0 and the concentration of H2O2 was obtained in the range of 0.02 – 20 mM H2O2, as is shown in the inset of Fig. 4. The detection limit (LOD), calculated following the 3σ IUPAC criterion, was 5 µM of H2O2. The repeatability expressed as the relative standard deviation, was 3.8% (N=7), whereas the reproducibility (established from three consecutive days) was 6.0%. A comparison of the analytical performance obtained with our fluorescent assay and other approaches for the detection of H2O2 in lens cleaning solutions is presented in Table 1. Figures of merit are comparable or even better than those of other enzymatic and non-enzymatic methods. Our fluorescent nanoprobe provides similar or even better LOD than most of methods included in Table 1. The dynamic range allows the detection of H2O2 in a wide concentration range being adequate for H2O2 detection in contact lens cleaning solutions, which typically contain H2O2 concentrations lower than 0.9 M. The high sample throughput (720 h-1) should be highlighted, which is highly desirable for routine analysis, being ~25-fold faster than those presented so far for contact lens cleaning solutions analysis. It should be mentioned that our CDs-based assay does not require the use of enzymes or expensive reagents, which considerably reduces the analysis cost.
10
3.4. Analytical results for H2O2 detection In order to demonstrate the accuracy of our fluorescent nanoprobe, several recovery studies were performed using different real samples. Three different contacts lens cleaning solutions purchased from a local optician´s shop were analyzed. The analytical results for sample solutions spiked with different amounts of H2O2 and diluted 5-fold show recoveries in the range of 95-105 % (Table S-2). Validation of the fluorescent assay was performed by comparing results with those obtained by the classical potassium permanganate method [36]. As is shown in Table S3, analytical results obtained with our assay were in good agreement with those obtained by classical titration method. In addition, analytical results were statistically compared for testing trueness. The t-test study confirmed that there were no significant differences between the results obtained by both methods for a confident level of 95 % (tcalculated= 2.42, ttabulated= 2.78 for sample 1 and tcalculated= 0.67, ttabulated= 2.78 for sample 2; n=3). These results indicate that the proposed method is reliable for H2O2 detection in contact lens cleaning solutions. 3.5. Antioxidant detection It is well known that the consumption of foods containing antioxidants is the most convenient way to reduce the effects of reactive oxygen species (ROS), thereby preventing health risks such as cell ageing and cancerous tumor growth, among others. In this work, ascorbic acid and glutathione were tested as antioxidant compounds in order to evaluate the potential of our sensing approach. To this end, known amounts of ascorbic acid were added to the mixture of sucrose/PEG/NaOH/H2O2 prior to UV irradiation. It was found that the presence of ascorbic acid during the synthesis causes 11
the fluorescence quenching of CDs, so ascorbic acid can be detected through a turn onoff sensing strategy. The fluorescence turn on-off can be explained from the ability of antioxidants to scavenge OH radicals generated upon photolysis of H2O2. Our optical nanoprobe provides a LOD of 6 µM ascorbic acid, which is suitable for its detection in fruits and pharmaceutical products. The equation for the calibration function was I0/I = 2185.5·CAA + 0.996 with a regression coefficient of 0.99. Glutathione caused the quenching of CDs fluorescence in a similar way. 3.6. Characterization of photogenerated fluorescent CDs As is shown from TEM images (Fig. 5A), CDs obtained by photochemical synthesis are monodisperse with an average size of 8 nm. A relevant factor influencing the size of CDs-PEG is the irradiation time. TEM images revealed that the application of longer irradiation times results in the generation of smaller CDs. CDs with average sizes of 6 and 3 nm were obtained after an UV irradiation time of 3 and 5 min, respectively. UV-vis absorption spectra of photogenerated CDs revealed a new band centered at 268 nm, hence suggesting that CDs are formed due to structural changes of sucrose. It has been reported that UV irradiation causes the photodegradation of carbohydrates in alkaline medium to form carbonyl-containing compounds, which can be monitored by UV absorption measurement at 268 nm [27]. In order to study the composition of CDs surface, FT-IR measurements were performed. Fig. 5(B) shows IR spectra obtained from different species, where a and b curves correspond to sucrose and PEG spectra, respectively, whereas c and d curves are CDs spectra synthesized in the absence and the presence of PEG, respectively. The sharp band at ~3350 cm-1 corresponds to OH stretching vibrations, whereas the peak centered 12
at ~2900 cm-1 indicates the presence of sp3 CH of alkane groups. In addition, a new peak at ~1640 cm-1 appears after UV irradiation for both CDs synthesized in the presence and absence of PEG, which seems to indicate that carbonyl groups from sucrose are formed. These data demonstrate that the photogenerated CDs are surrounded by hydrophilic groups, which lead to high aqueous solubility. Photogenerated CDs showed a blue color under UV irradiation (365 nm) that is easily visible to the naked eye. It was observed that the fluorescence emission depends on the excitation wavelength, as is shown in Fig. S-2. The emission band of CDs shifted from 420 to 520 nm when CDs were excited at a wavelength ranging from 300 to 500 nm. Additionally, CDs show up-conversion fluorescence properties, so low-energy light (NIR or IR) is converted to higher-energy light (UV or visible). CDs excited by long wavelength light (700-900 nm) showed up-converted emissions located in the range of 435-500 nm. This may be due to a multiphoton process, which leads to anti Stokes type emission [5]. Photochemical synthesis of CDs has several advantages as compared to other methods described in the literature (Table 2). Among the features shown in the table, the fast reaction rates and the soft reaction conditions (i.e., room temperature) should be highlighted. Moreover, up-conversion fluorescence is an attractive optical property of as-prepared CDs, which enables many promising applications, especially in the biological field due to the use of low energy radiations as excitation sources in fluorescent assays [5]. In addition, the integration of synthesis and sensing events within one only step allows building a fast fluorescent assay since subsequent purification steps of synthesized CDs are avoided. The latter operation usually takes from some
13
hours to several days. Ultimately, a high reproducibility (i.e., variation of ±2% in the intrinsic fluorescence of CDs) of the synthesis approach should be highlighted. 3.7. Sensing mechanism for H2O2 detection In order to explain the fluorescence enhancement observed when CDs are synthesized in the presence of H2O2, two possible mechanisms are proposed. On the one hand, when H2O2 is added to the synthesis medium, the CDs size is reduced 2-fold (Fig. 5C). As was mentioned before, the use of longer UV irradiation times (without addition of H2O2) also causes a decrease in the CDs size as a result of photooxidation. Thus, CDs sizes of 8, 6 and 3 nm were obtained after UV irradiation for 1, 3 and 5 min, respectively. Therefore, highly oxidant OH radicals formed upon photolysis of H2O2. OH radicals could speed up the photooxidation of carbohydrates and thus, smaller CDs are obtained within a short reaction time. The decrease in CDs size would cause a higher quantum confinement of emissive energy traps resulting in enhanced fluorescence [49, 50]. On the other hand, OH radicals could cause the photochemical etching of CDs-PEG, thus yielding surface defects responsible for the fluorescence enhancement observed [5]. Both mechanisms are supported by experiments carried out with antioxidants. The presence of antioxidants such as ascorbic acid or glutathione prevents the acceleration of both the photochemical reaction and photochemical etching since antioxidants behave as OH radical scavengers.
14
4. Conclusions The potential of the photochemical synthesis of CDs was clearly demonstrated. The integration of CDs synthesis and sensing for detection of H2O2 and antioxidants allows expanding the use of fluorescent CDs for analytical applications. The mechanism involved in H2O2 sensing was studied by UV-Vis absorption, fluorescence, TEM and FT-IR measurements, revealing that the decrease in CDs size is the responsible for the fluorescence enhancement (turn on) due to acceleration of the photochemical reaction caused by the presence of OH radicals. This process can be efficiently inhibited by antioxidants such as ascorbic acid or glutathione, which allows building a turn on-off system. To sum up, the new approach for CDs fabrication exhibits the following advantages: i) reduced time for synthesis and sensing due to the integration of both processes; ii) increased greenness as compared to conventional synthesis pathways due to the use of UV irradiation as energy source, thus allowing soft synthesis conditions; iii) synthesis of CDs with up-conversion fluorescence properties, which is convenient for future biological applications; iv) minimal consumption of sample and reagents; v) high sample throughput; vi) ability of field analysis due to the use of a portable microfluorospectrometer. Acknowledgements Financial support from the Spanish Ministry of Economy and Competitiveness (Project CTQ2012-32788) and the European Commission (FEDER) is gratefully acknowledged. V. Romero thanks the Spanish Ministry of Education, Culture and Sports, for predoctoral research grants. E. López (CACTI, University of Vigo) is gratefully 15
acknowledged for performing the FT-IR measurements.
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D. Harms, R. Than, U. Pinkernell, M. Schmidt, B. Krebs, U. Karst, Selective determination of hydrogen peroxide in household products using its molecular recognition by a dinuclear iron(iii) complex, Analyst 123 (1998) 2323-2327.
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Council of Europe, Eurepean Pharmacopoeia, 5th ed., 2004.
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J.-M. Liu, L.-p. Lin, X.-X. Wang, S.-Q. Lin, W.-L. Cai, L.-H. Zhang, Z.-Y. Zheng, Highly selective and sensitive detection of Cu2+ with lysine enhancing bovine serum albumin modified-carbon dots fluorescent probe, Analyst 137 (2012) 2637-2642.
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Z. Lin, W. Xue, H. Chen, J.-M. Lin, Peroxynitrous-acid-induced chemiluminescence of fluorescent carbon dots for nitrite sensing, Anal. Chem. 83 (2011) 8245-8251.
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S. Barman, M. Sadhukhan, Facile bulk production of highly blue fluorescent graphitic carbon nitride quantum dots and their application as highly selective and sensitive sensors for the detection of mercuric and iodide ions in aqueous media, J. Mater. Chem. 22 (2012) 21832-21837.
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A. Zhao, C. Zhao, M. Li, J. Ren, X. Qu, Ionic liquids as precursors for highly luminescent, surface-different nitrogen-doped carbon dots used for label-free detection of Cu2+/Fe3+ and cell imaging, Anal. Chim. Acta 809 (2014) 128-133.
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Z. Ma, H. Ming, H. Huang, Y. Liu, Z. Kang, One-step ultrasonic synthesis of fluorescent N-doped carbon dots from glucose and their visible-light sensitive photocatalytic ability, New J. Chem. 36 (2012) 861-864.
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H. Li, X. He, Y. Liu, H. Yu, Z. Kang, S.-T. Lee, Synthesis of fluorescent carbon nanoparticles directly from active carbon via a one-step ultrasonic treatment, Mat. Res. Bull. 46 (2011) 147-151.
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S.S. Wee, Y.H. Ng, S.M. Ng, Synthesis of fluorescent carbon dots via simple acid hydrolysis of bovine serum albumin and its potential as sensitive sensing probe for lead(II) ions, Talanta 116 (2013) 71-76.
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Y. Liu, C.-y. Liu, Z.-y. Zhang, Synthesis of highly luminescent graphitized carbon dots and the application in the Hg2+ detection, Appl. Surf. Sci. 263 (2012) 481-485.
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K. Linehan, H. Doyle, Size controlled synthesis of carbon quantum dots using hydride reducing agents, J. Mater. Chem. 2 (2014) 6025-6031.
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Figure captions Figure 1: Effect of the carbon sources used as precursors of CDs. The solid and dotted lines represent the analytical response (I/I0) and fluorescence intensity (relative fluorescence units, RFU) of CDs, respectively. Experimental conditions: precursor mass, 30 mg; sample volume, 1.3 mL; H2O2 concentration, 2 mM; PEG volume, 1.2 mL; NaOH concentration, 3.5 M; NaOH volume, 200 µL. Figure 2: (A) Effect of sucrose mass (experimental conditions: sample volume, 1.3 mL; H2O2 concentration, 2 mM; PEG volume, 1.2 mL; NaOH concentration, 3.5 M; NaOH volume, 200 µL). (B) Effect of stabilizing agent on the intrinsic fluorescence of CDs (experimental conditions: sucrose mass, 20 mg; sample volume, 1.5 mL; stabilizing agent volume, 1.2 mL; NaOH concentration, 3.5 M; NaOH volume, 200 µL; irradiation time, 2 min). (C) Analytical response obtained by using different concentrations of stabilizing agents (experimental conditions: sucrose mass, 20 mg; sample volume, 1.5 mL; H2O2 concentration, 2 mM; NaOH concentration, 3.5 M; NaOH volume, 200 µL; irradiation time, 2 min). The solid and dotted lines represent the analytical response (I/I0) and fluorescence intensity (relative fluorescence units, RFU) of CDs, respectively. Figure 3: (A) Effect of NaOH concentration (experimental conditions: sucrose mass, 20 mg; sample volume, 1.5 mL; H2O2 concentration, 2 mM; PEG volume, 0.8 mL; NaOH 20
volume, 200 µL; irradiation time, 2 min). (B) Effect of irradiation time (experimental conditions: precursor mass, 20 mg; sample volume, 1.5 mL; H2O2 concentration, 2 mM; PEG volume, 0.8 mL; NaOH concentration, 3 M; NaOH volume, 200 µL). The solid and dotted lines represent the analytical response (I/I0) and fluorescence intensity (relative fluorescence units, RFU) of CDs, respectively. Figure 4: Fluorescence spectra of photogenerated CDs in the presence of different amounts of H2O2 and representation of calibration curve. Experimental conditions: sucrose mass, 20 mg; sample volume, 1.5 mL; H2O2 concentration, 0.02–20 mM; PEG volume, 0.8 mL; NaOH concentration, 3 M; NaOH volume, 200 µL; irradiation time, 1 min. Figure 5: (A) TEM image of CDs prepared in the absence of H2O2; (B) FT-IR spectra for the different studied samples; (C) TEM image of CDs prepared in the presence of H2O2 (2 mM concentration). Experimental conditions for TEM images: sucrose mass, 20 mg; sample volume, 1.5 mL; PEG volume, 0.8 mL; NaOH concentration, 3 M; NaOH volume, 200 µL; irradiation time, 1 min.
Table 1: Comparison of some analytical characteristics for the detection of H2O2 in lens cleaning solutions by different methods. Sample a
Method
Linear range
LOD (µM)
Repeatability (%)
throughput (h-1)
Ref.
Enzymatic methods Amperometry
0.2-3.4 mM
40
2.4
-
[31]
Chemiluminescence
0.1-3.0
670
7.1
12
[32]
21
mM 0.01-0.1 mM
5.9
-
45
[33]
1.3-10 mM
500
-
45
[33]
9.0-130 µM
3
1
23
[34]
SI-LOV-spectrophotometry
FI-spectrophotometry
Non-enzymatic methods 0.02-1.0 mM
2.05
-
-
[14]
2.0-80 mM
19.6
-
-
[14]
5-35000 µM
1.6
3.9
45-610 µM
28
1.9
26
[34]
100-4500 µM
80
1.0
40
[34]
50-500 µM
30
1.5
22
[34]
30-615 µM
22
1
26
34]
Spectrofluorimetry
0.7-10 mM
350
-
54
[17]
Spectrophotometry
9.0-300 µM
3
-
12
[35]
20-20000 µM
5
3.8
720
This work
Amperometry
[15]
FI-spectrophotometry
CDs nanoprobemicrofluorospectrometry a
Abbreviations: FI, flow injection; LOV, lab-on-valve; SI, sequential injection.
22
Table 2: Comparison of the main characteristics of several synthesis procedures for CDs.
Synthetic approach
Microwave treatment
Ultrasonic treatment
Hydrotherm al method
Precursors
CD s size (n m)
Time for synthes is
Temperat ure for synthesis (oC)
Dialys is
Upconversi on properties
Citric acid/EDA
5
2 min
-
YES
NO
Sucrose/H2SO4
3-6
18 min
-
YES
-
Glucose/PEG
5
2 min
-
YES
-
[39]
Glycerine/PEG/se rine
3-4
10 min
-
YES
NO
[40]
Formamide
215
30 min
-
NO
NO
Ionic liquids
2-6
12 min
-
YES
NO
Glucose/NH4OH
10
24 h
Room temperatur e
YES
YES
Active carbon/H2O2
510
15 h
Room temperatur e
NO
YES
Fructose/PEG/Na OH
2.5
1 min
Room temperatur e
NO
-
BSA/H2SO4
1-2
2h
50
YES
-
EG/H2SO4
1-4
8h
140
NO
NO
Glycerol/PEGNH2
5.5
1h
230
YES
NO
Pepper
2-7
5h
180
YES
YES
Folic acid/EG
4.5
12 h
180
YES
NO
Pomelo peel
2-4
51 h
200
NO
-
[6]
Watermelon peel
2
2.5 h
220
YES
NO
[7]
23
Ref.
[37] [38]
[41] [42]
[43]
[44]
[11]
[45] [46] [47] [9] [48]
Photochemi cal treatment
Banana peel
5.5
3h
200
NO
-
[8]
Bamboo leave
3.6
6h
200
NO
NO
[10]
1 min
Room temperatu re
YES
Thi s wor k
sucrose/PEG/Na OH
8
NO
BSA, bovine serum albumin; EDA, ethylenediamine; EG, ethylene glycol; PVP, polyvinylpyrrolidone.
Highlights · · ·
Photochemical synthesis of fluorescent CDs is described for the first time. Integration of CDs synthesis and sensing within a single step is achieved. Turn on (H2O2) and turn on-off (antioxidants) fluorescent nanoprobes are obtained.
24
*Graphical Abstract (for review)
Figure
Figure
Figure
Figure
Figure
Figure
Figure
PII: DOI: Reference:
S0039-9140(15)30220-4 http://dx.doi.org/10.1016/j.talanta.2015.07.093 TAL15858
To appear in: Talanta Received date: 10 June 2015 Revised date: 27 July 2015 Accepted date: 31 July 2015 Cite this article as: Isabel Costas-Mora, Vanesa Romero, Isela Lavilla and Carlos Bendicho, In situ photochemical synthesis of fluorescent carbon dots for optical sensing of hydrogen peroxide and antioxidants, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.07.093 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.
In situ photochemical synthesis of fluorescent carbon dots for optical sensing of hydrogen peroxide and antioxidants Isabel Costas-Mora, Vanesa Romero, Isela Lavilla and Carlos Bendicho* Departamento de Química Analítica y Alimentaria, Área de Química Analítica, Facultad de Química, Universidad de Vigo, Campus As Lagoas-Marcosende s/n, 36310 Vigo, Spain. *Corresponding author. Tel.: +34-986-812281; fax: +34-986-812556; e-mail: [email protected] Abstract A new synthesis approach for obtaining fluorescent carbon dots (CDs) based on UV irradiation of carbohydrates was developed. The photochemical synthesis pathway allows the formation of water soluble CDs of analytical usefulness within one min. CDs obtained by photochemical treatment from the sucrose/NaOH/poly(ethylene glycol) system are monodisperse with an average size of 8 nm as determined by transmission electron microscopy. A dramatic increase in the CDs fluorescence (turn on) is observed when H2O2 is added. The decrease in CDs size occurring by the action of highly oxidant OH radicals gives rise to confinement of emissive energy traps and, in turn, to fluorescence enhancement. Antioxidants such as ascorbic acid and glutathione inhibit the photochemical reaction giving rise to a decrease in fluorescence of the CDs/H2O2 system (turn on-off). The detection limit was 5 µM H2O2 and the repeatability expressed as the relative standard deviation was 3.8% (N=7). The photochemical synthesis of CDs allows building a green, low-cost, safe and fast assay for the detection of H2O2 and 1
antioxidants. An application of the novel fluorescent nanoprobe to H2O2 detection in contact lens cleaning solutions is performed. Keywords: carbon dots; fluorescence; nanoprobe; photochemical synthesis; hydrogen peroxide; antioxidants.
1. Introduction Fluorescent nanoparticles (NPs) have been increasingly used as optical probes for the detection of a wide variety of chemical species [1-3]. In the last years, carbon dots (CDs) have emerged as one of the most interesting optical nanoprobes due to their high aqueous stability, easy functionalization and low toxicity [4]. CDs are fluorescent nanoparticles with sizes comprised between 2-10 nm, which can be synthesized from natural and non-toxic precursors such as carbohydrates. So far, synthesis pathways for obtaining fluorescent CDs include laser ablation, arcdischarge, electrochemical exfoliation, microwave treatment, thermal carbonization, acid dehydration, etc. [4, 5]. However, most synthesis methods have some drawbacks such as long operation time and harsh synthesis conditions, i.e. high temperature, so new approaches should be investigated in order to develop greener synthesis methods. Some studies have been focused on the use of greener carbon sources as CDs precursors such as pomelo, watermelon and banana peels [6-8], pepper [9] or bamboo leaves [10], which cause a low environmental impact and are cost-effective, yet the reaction time and temperature used to obtain fluorescent CDs remain a drawback. Generally, fluorescent NPs-based assays described in the literature for sensing different chemical species require two-step, i.e., firstly fluorescent NPs are synthesized following 2
different procedures, and secondly, a recognition event is needed for detection of the target analyte. Recently, our research group has developed a new detection strategy for methylmercury based on the integration of sonochemical synthesis of CDs and sensing within a single step [11]. This approach eliminates the need for large and tedious procedures for purification and stabilization of freshly synthesized CDs, and moreover, the time required to accomplish the assay is remarkably shortened. H2O2 is a highly reactive oxidant often used in water treatment plants as well as in household products due to its antimicrobial properties. Its use is especially relevant in contact lens cleaning solutions in order to eliminate pathogens by oxidative process. Nevertheless, H2O2 levels need to be controlled in those samples, since H2O2 can be harmful to the ocular epithelium and cornea causing eyes irritation and possible corneal damage [12]. To date, most of the H2O2 detection methods are based on the use of enzymes, which pose some limitations such as high cost and stability problems due to changes in temperature, pH and the presence of concomitants. In recent years, an increasing number of nanostructured materials have been implemented in nonenzymatic assays for H2O2 detection. Thus, nanocomposites [13, 14], metallic nanoparticles [15], inorganic nanoparticles [16] and QDs [17], have been used for monitoring H2O2 in household products and contact lens cleaning solutions. Although the CDs ability to detect H2O2 has already been reported [18-22], those methods were mainly applied to detect H2O2 in biological matrices such as live cells, zebrafish larvae, serum and saliva [20-22]. So far, no application has appeared for the determination of H2O2 in household products and contact lens cleaning solutions. In this work, synthesis of fluorescent CDs upon application of UV radiation to carbohydrates is demonstrated for the first time. Enhancement/quenching effects of the
3
CDs fluorescence caused by the presence of H2O2 and antioxidants (e.g., ascorbic acid and glutathione) in the reaction medium can be the basis for a fast, low cost and sensitive fluorescent assay, which integrates synthesis and sensing in one step. Application of the novel assay for the detection of H2O2 in contact lens cleaning solutions is provided. 2. Experimental Section 2.1. Reagents and Chemicals Ultrapure water obtained from an Ultra Clear TWF EDI UV TM system (Siemens AG, Barsbuettel, Germany) was used for the preparation of working solutions. D-glucose, D-fructose, sucrose, (Sigma-Aldrich, St. Louis, MO, USA) and starch (Probus, Badalona, Barcelona) were tested as carbon sources. Several species purchased from Sigma-Aldrich were tried as stabilizing agents including poly(ethylene glycol) (PEG, MW=200), Triton X-100,Triton X-114, Tween 80, polyethylenimine (PEI), hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), polyvinylpyrrolidone (PVP), bovine serum albumin (BSA), Lcysteine, glutathione (GSH) and citric acid. NaOH was obtained from Prolabo (Paris, France). Potassium permanganate (Carlo Erba, Italy) and sulfuric acid (Prolabo) were used in order to validate the reliability of the fluorescent assay. The following compounds were tested as potential interferents: NaCl, sodium tetraborate,
sodium
citrate,
calcium
4
carbonate
(Sigma-Aldrich),
ethylenediaminetetraacetic acid (EDTA) (Prolabo), sodium phosphate (Panreac, Barcelona, Spain). 2.2. Apparatus Photochemical treatments were carried out in open quartz tubes (15 mm i.d., 125 mm height) using a 705 UV digester (Metrohm) equipped with a high-pressure mercury lamp (500 W). In addition, it is equipped with a holder for twelve tubes. A Thermo Scientific NanoDrop 3300 Fluorospectrometer whose technical specifications and operation mode have been outlined in earlier works [23, 24] was used to carry out fluorescence measurements. The fluorescence intensity at 517 nm was measured after excitation at a wavelength of 470 nm using blue LED. It should be mentioned that this miniaturized instrument can operate through connection to a computer via USB, thus facilitating field analysis. The effect of the excitation wavelength over the emission wavelength was studied using a Shimadzu RF-1501 Spectrofluorometer. In order to characterize CDs synthesized by photochemical treatment, UV-Vis absorption
measurements
were
performed
using
a
NanoDrop
ND-1000
spectrophotometer whose operation mode was described in an earlier work [25]. Transmission electron microscopy (TEM) was performed with JEOL JEM-1010 microscope operating at an acceleration voltage of 100 kV. TEM experiments were made by dropping the sample onto a carbon-coated copper grid. Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet 6700 spectrometer within the range of 400-4000 cm−1 with a resolution of 4 cm−1.
5
2.3 Experimental procedure for building the fluorescent nanoprobe Sucrose (20 mg) was introduced in a quartz tube and then, 1.5 mL of aqueous sample containing H2O2 was added. Once sucrose was solubilized, 0.8 mL of PEG and 200 µL of NaOH 3 M were added to the mixture. After that, the tubes were introduced in the sample holder of the UV digester and irradiated for 1 min. The color of the bulk solution changed from clear to light yellow. Finally, a 2 μL aliquot of the sample was placed onto the pedestal of the microfluorospectrometer in order to measure the fluorescence signal. A blank experiment was performed before each measurement in order to calculate analytical response (I/I0, where I0 and I represents the fluorescence intensity of CDs synthesized in the absence and in the presence of H2O2, respectively).
3. Results and discussion 3.1. Optimization of experimental parameters influencing the fluorescent assay In order to assess the effects of the different variables involved in the proposed system for H2O2 detection, the 2 factorial fractional design was applied for screening optimization [26]. This design allows assessing the effect of 4 variables (i.e. sucrose mass, PEG concentration, NaOH concentration and UV irradiation time) by performing 8 experiments in duplicate. The significance of the studied variables was established by comparison with the experimental error, calculated as two times the average standard deviation of all experiments (2 s̅ ). Standardized effects showed in Fig. S-1 reveal that all studied variables had a significant effect, so the assay was optimized in more detail following the univariate approach. 6
3.1.1. Selection of the carbon source and its concentration Carbohydrates have proved to be suitable precursors for building CDs following different synthetic routes. In studies concerning the removal of carbohydrates by UV irradiation, it has been shown that photodegradation occurs in alkaline medium [27]. In this work, different carbohydrates were tested as CDs precursors including D-glucose, D-fructose, sucrose and starch. It was observed that CDs can be synthesized from different carbon precursors using different irradiation times. The results obtained with 30 mg of each carbohydrate are shown in Fig. 1. As can be observed (intrinsic fluorescence signal, dotted line), fluorescent CDs can be synthesized using any of the tested carbohydrates, but different reaction kinetics were observed with each carbon precursor. Therefore, the use of simple sugars such as glucose and fructose allows synthesizing CDs in very short irradiation times, i.e. 45 and 30 s, respectively. The use of sucrose or starch requires longer irradiation times (i.e. 60 s) to achieve fluorescent CDs. The behavior of starch can be ascribed to the large number of glucose units of this polysaccharide. Sucrose is a disaccharide composed of glucose and fructose units, so higher energy is required to break down its structure, and hence, a longer irradiation time is required. Given that all carbohydrates tried lead to a rapid synthesis of fluorescent CDs, all of them were tested as fluorescent nanoprobes for H2O2 detection. As is shown in Fig. 1 (analytical response, solid line), the I/I0 ratio depends on both the carbon source and irradiation time. In all cases, a remarkable increase in CDs fluorescence is observed in the presence of H2O2. For glucose, fructose and starch, a maximum analytical response close to I/I0~1.5 is obtained in a short time (i.e., 30-90 s). Nevertheless, the use of sucrose as CDs precursor allows achieving a significant improvement of the analytical 7
response (I/I0~2) using an irradiation time of 2 min. In view of the obtained results, sucrose was selected as carbon source and its concentration was optimized in order to reach an adequate sensitivity for H2O2 detection. The effect of sucrose mass was studied in the range of 5-60 mg. Fig. 2(A) shows both the intrinsic fluorescence intensity of CDs and analytical responses for H2O2 detection. As can be observed, the best analytical response is obtained using a sucrose mass in the range of 20-25 mg. 3.1.2. Selection of the stabilizing agent and its concentration Stabilizing agents may influence the optical properties of CDs [28, 29]. For this reason, several stabilizing agents including PEG, triton X-100, triton X-114, tween 80, PEI, CTAB, SDS, PVP, BSA, L-cysteine, GSH and citric acid were tested to obtain suitable fluorescent CDs for the detection of H2O2. As is shown in Fig. 2(B), the addition of PEG, tween 80 or BSA prior to UV irradiation allows obtaining CDs with increased fluorescence as compared to CDs synthesized in the absence of stabilizing agent. For this reason, different concentrations of stabilizing agents (PEG, tween 80 and BSA) were tried for the detection of H2O2. As is shown in Fig. 2(C), an optimal analytical response for H2O2 was only obtained when PEG was added to the reaction medium. An increase in the PEG concentration up to 30 % (v/v) led to an increased analytical signal. Therefore, a PEG concentration of 35 % (v/v) was chosen for further experiments. 3.1.3. Effect of NaOH concentration There are several studies pointing out that pH is a critical parameter in order to get fluorescent CDs [10, 11, 30]. In this work, it was observed that alkaline medium is mandatory in order to obtain fluorescent CDs, since photodegradation of carbohydrates requires an alkaline medium [27]. For this reason, the effect of NaOH concentration
8
added was studied in the range of 1–6 M (Fig. 3A). A NaOH concentration of 3 M was chosen in order to reach the best analytical response. 3.1.4. Effect of UV irradiation time Irradiation times ranging from 45 to 210 s were applied. As is shown in Fig. 3(B), an increased analytical response for H2O2 is obtained using a short irradiation time. An irradiation time longer than 120 s causes the analytical signal to decrease. In view of the obtained results, a time of one min was selected as optimum irradiation time. These conditions allow not only the sensitive detection of H2O2 but also a high sample throughput, since 12 samples can be run per min. 3.2. Study of potential interferences In order to investigate the effect of potential matrix interferences in the sample, the fluorescence enhancement caused by 1 mM of H2O2 in the presence of several foreign substances was assessed. An interference effect was considered significant when the analytical response varied beyond ±10%. Several compounds commonly present in contact lens cleaning solutions were evaluated as potential interferences. Analytical results for interference studies are shown in Table S-1. Depressive effects on the analytical response were observed for sodium citrate, sodium tetraborate, EDTA and sodium phosphate at a concentration higher than 25, 3, 0.1 and 3.5% (m/v), respectively. On the other hand, it was found that sodium chloride caused a positive effect at a concentration higher than 7% (m/v). Carbonate was also tested as a potential interference because it is a potent OH radical scavenger. It was observed that carbonate concentrations higher than 0.3 M caused a decreased analytical
9
signal. It must be highlighted that the tolerance limits found in the interference study are higher than the concentrations usually present in contact lens cleaning solutions. 3.3. Analytical characteristics of the fluorescent nanoprobe Analytical figures of merit of the fluorescent assay were established under optimal conditions. When CDs synthesis is performed in the presence of H2O2, a significant fluorescence enhancement occurs and no spectral shift of the emission band is observed. A linear relationship between I/I0 and the concentration of H2O2 was obtained in the range of 0.02 – 20 mM H2O2, as is shown in the inset of Fig. 4. The detection limit (LOD), calculated following the 3σ IUPAC criterion, was 5 µM of H2O2. The repeatability expressed as the relative standard deviation, was 3.8% (N=7), whereas the reproducibility (established from three consecutive days) was 6.0%. A comparison of the analytical performance obtained with our fluorescent assay and other approaches for the detection of H2O2 in lens cleaning solutions is presented in Table 1. Figures of merit are comparable or even better than those of other enzymatic and non-enzymatic methods. Our fluorescent nanoprobe provides similar or even better LOD than most of methods included in Table 1. The dynamic range allows the detection of H2O2 in a wide concentration range being adequate for H2O2 detection in contact lens cleaning solutions, which typically contain H2O2 concentrations lower than 0.9 M. The high sample throughput (720 h-1) should be highlighted, which is highly desirable for routine analysis, being ~25-fold faster than those presented so far for contact lens cleaning solutions analysis. It should be mentioned that our CDs-based assay does not require the use of enzymes or expensive reagents, which considerably reduces the analysis cost.
10
3.4. Analytical results for H2O2 detection In order to demonstrate the accuracy of our fluorescent nanoprobe, several recovery studies were performed using different real samples. Three different contacts lens cleaning solutions purchased from a local optician´s shop were analyzed. The analytical results for sample solutions spiked with different amounts of H2O2 and diluted 5-fold show recoveries in the range of 95-105 % (Table S-2). Validation of the fluorescent assay was performed by comparing results with those obtained by the classical potassium permanganate method [36]. As is shown in Table S3, analytical results obtained with our assay were in good agreement with those obtained by classical titration method. In addition, analytical results were statistically compared for testing trueness. The t-test study confirmed that there were no significant differences between the results obtained by both methods for a confident level of 95 % (tcalculated= 2.42, ttabulated= 2.78 for sample 1 and tcalculated= 0.67, ttabulated= 2.78 for sample 2; n=3). These results indicate that the proposed method is reliable for H2O2 detection in contact lens cleaning solutions. 3.5. Antioxidant detection It is well known that the consumption of foods containing antioxidants is the most convenient way to reduce the effects of reactive oxygen species (ROS), thereby preventing health risks such as cell ageing and cancerous tumor growth, among others. In this work, ascorbic acid and glutathione were tested as antioxidant compounds in order to evaluate the potential of our sensing approach. To this end, known amounts of ascorbic acid were added to the mixture of sucrose/PEG/NaOH/H2O2 prior to UV irradiation. It was found that the presence of ascorbic acid during the synthesis causes 11
the fluorescence quenching of CDs, so ascorbic acid can be detected through a turn onoff sensing strategy. The fluorescence turn on-off can be explained from the ability of antioxidants to scavenge OH radicals generated upon photolysis of H2O2. Our optical nanoprobe provides a LOD of 6 µM ascorbic acid, which is suitable for its detection in fruits and pharmaceutical products. The equation for the calibration function was I0/I = 2185.5·CAA + 0.996 with a regression coefficient of 0.99. Glutathione caused the quenching of CDs fluorescence in a similar way. 3.6. Characterization of photogenerated fluorescent CDs As is shown from TEM images (Fig. 5A), CDs obtained by photochemical synthesis are monodisperse with an average size of 8 nm. A relevant factor influencing the size of CDs-PEG is the irradiation time. TEM images revealed that the application of longer irradiation times results in the generation of smaller CDs. CDs with average sizes of 6 and 3 nm were obtained after an UV irradiation time of 3 and 5 min, respectively. UV-vis absorption spectra of photogenerated CDs revealed a new band centered at 268 nm, hence suggesting that CDs are formed due to structural changes of sucrose. It has been reported that UV irradiation causes the photodegradation of carbohydrates in alkaline medium to form carbonyl-containing compounds, which can be monitored by UV absorption measurement at 268 nm [27]. In order to study the composition of CDs surface, FT-IR measurements were performed. Fig. 5(B) shows IR spectra obtained from different species, where a and b curves correspond to sucrose and PEG spectra, respectively, whereas c and d curves are CDs spectra synthesized in the absence and the presence of PEG, respectively. The sharp band at ~3350 cm-1 corresponds to OH stretching vibrations, whereas the peak centered 12
at ~2900 cm-1 indicates the presence of sp3 CH of alkane groups. In addition, a new peak at ~1640 cm-1 appears after UV irradiation for both CDs synthesized in the presence and absence of PEG, which seems to indicate that carbonyl groups from sucrose are formed. These data demonstrate that the photogenerated CDs are surrounded by hydrophilic groups, which lead to high aqueous solubility. Photogenerated CDs showed a blue color under UV irradiation (365 nm) that is easily visible to the naked eye. It was observed that the fluorescence emission depends on the excitation wavelength, as is shown in Fig. S-2. The emission band of CDs shifted from 420 to 520 nm when CDs were excited at a wavelength ranging from 300 to 500 nm. Additionally, CDs show up-conversion fluorescence properties, so low-energy light (NIR or IR) is converted to higher-energy light (UV or visible). CDs excited by long wavelength light (700-900 nm) showed up-converted emissions located in the range of 435-500 nm. This may be due to a multiphoton process, which leads to anti Stokes type emission [5]. Photochemical synthesis of CDs has several advantages as compared to other methods described in the literature (Table 2). Among the features shown in the table, the fast reaction rates and the soft reaction conditions (i.e., room temperature) should be highlighted. Moreover, up-conversion fluorescence is an attractive optical property of as-prepared CDs, which enables many promising applications, especially in the biological field due to the use of low energy radiations as excitation sources in fluorescent assays [5]. In addition, the integration of synthesis and sensing events within one only step allows building a fast fluorescent assay since subsequent purification steps of synthesized CDs are avoided. The latter operation usually takes from some
13
hours to several days. Ultimately, a high reproducibility (i.e., variation of ±2% in the intrinsic fluorescence of CDs) of the synthesis approach should be highlighted. 3.7. Sensing mechanism for H2O2 detection In order to explain the fluorescence enhancement observed when CDs are synthesized in the presence of H2O2, two possible mechanisms are proposed. On the one hand, when H2O2 is added to the synthesis medium, the CDs size is reduced 2-fold (Fig. 5C). As was mentioned before, the use of longer UV irradiation times (without addition of H2O2) also causes a decrease in the CDs size as a result of photooxidation. Thus, CDs sizes of 8, 6 and 3 nm were obtained after UV irradiation for 1, 3 and 5 min, respectively. Therefore, highly oxidant OH radicals formed upon photolysis of H2O2. OH radicals could speed up the photooxidation of carbohydrates and thus, smaller CDs are obtained within a short reaction time. The decrease in CDs size would cause a higher quantum confinement of emissive energy traps resulting in enhanced fluorescence [49, 50]. On the other hand, OH radicals could cause the photochemical etching of CDs-PEG, thus yielding surface defects responsible for the fluorescence enhancement observed [5]. Both mechanisms are supported by experiments carried out with antioxidants. The presence of antioxidants such as ascorbic acid or glutathione prevents the acceleration of both the photochemical reaction and photochemical etching since antioxidants behave as OH radical scavengers.
14
4. Conclusions The potential of the photochemical synthesis of CDs was clearly demonstrated. The integration of CDs synthesis and sensing for detection of H2O2 and antioxidants allows expanding the use of fluorescent CDs for analytical applications. The mechanism involved in H2O2 sensing was studied by UV-Vis absorption, fluorescence, TEM and FT-IR measurements, revealing that the decrease in CDs size is the responsible for the fluorescence enhancement (turn on) due to acceleration of the photochemical reaction caused by the presence of OH radicals. This process can be efficiently inhibited by antioxidants such as ascorbic acid or glutathione, which allows building a turn on-off system. To sum up, the new approach for CDs fabrication exhibits the following advantages: i) reduced time for synthesis and sensing due to the integration of both processes; ii) increased greenness as compared to conventional synthesis pathways due to the use of UV irradiation as energy source, thus allowing soft synthesis conditions; iii) synthesis of CDs with up-conversion fluorescence properties, which is convenient for future biological applications; iv) minimal consumption of sample and reagents; v) high sample throughput; vi) ability of field analysis due to the use of a portable microfluorospectrometer. Acknowledgements Financial support from the Spanish Ministry of Economy and Competitiveness (Project CTQ2012-32788) and the European Commission (FEDER) is gratefully acknowledged. V. Romero thanks the Spanish Ministry of Education, Culture and Sports, for predoctoral research grants. E. López (CACTI, University of Vigo) is gratefully 15
acknowledged for performing the FT-IR measurements.
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Figure captions Figure 1: Effect of the carbon sources used as precursors of CDs. The solid and dotted lines represent the analytical response (I/I0) and fluorescence intensity (relative fluorescence units, RFU) of CDs, respectively. Experimental conditions: precursor mass, 30 mg; sample volume, 1.3 mL; H2O2 concentration, 2 mM; PEG volume, 1.2 mL; NaOH concentration, 3.5 M; NaOH volume, 200 µL. Figure 2: (A) Effect of sucrose mass (experimental conditions: sample volume, 1.3 mL; H2O2 concentration, 2 mM; PEG volume, 1.2 mL; NaOH concentration, 3.5 M; NaOH volume, 200 µL). (B) Effect of stabilizing agent on the intrinsic fluorescence of CDs (experimental conditions: sucrose mass, 20 mg; sample volume, 1.5 mL; stabilizing agent volume, 1.2 mL; NaOH concentration, 3.5 M; NaOH volume, 200 µL; irradiation time, 2 min). (C) Analytical response obtained by using different concentrations of stabilizing agents (experimental conditions: sucrose mass, 20 mg; sample volume, 1.5 mL; H2O2 concentration, 2 mM; NaOH concentration, 3.5 M; NaOH volume, 200 µL; irradiation time, 2 min). The solid and dotted lines represent the analytical response (I/I0) and fluorescence intensity (relative fluorescence units, RFU) of CDs, respectively. Figure 3: (A) Effect of NaOH concentration (experimental conditions: sucrose mass, 20 mg; sample volume, 1.5 mL; H2O2 concentration, 2 mM; PEG volume, 0.8 mL; NaOH 20
volume, 200 µL; irradiation time, 2 min). (B) Effect of irradiation time (experimental conditions: precursor mass, 20 mg; sample volume, 1.5 mL; H2O2 concentration, 2 mM; PEG volume, 0.8 mL; NaOH concentration, 3 M; NaOH volume, 200 µL). The solid and dotted lines represent the analytical response (I/I0) and fluorescence intensity (relative fluorescence units, RFU) of CDs, respectively. Figure 4: Fluorescence spectra of photogenerated CDs in the presence of different amounts of H2O2 and representation of calibration curve. Experimental conditions: sucrose mass, 20 mg; sample volume, 1.5 mL; H2O2 concentration, 0.02–20 mM; PEG volume, 0.8 mL; NaOH concentration, 3 M; NaOH volume, 200 µL; irradiation time, 1 min. Figure 5: (A) TEM image of CDs prepared in the absence of H2O2; (B) FT-IR spectra for the different studied samples; (C) TEM image of CDs prepared in the presence of H2O2 (2 mM concentration). Experimental conditions for TEM images: sucrose mass, 20 mg; sample volume, 1.5 mL; PEG volume, 0.8 mL; NaOH concentration, 3 M; NaOH volume, 200 µL; irradiation time, 1 min.
Table 1: Comparison of some analytical characteristics for the detection of H2O2 in lens cleaning solutions by different methods. Sample a
Method
Linear range
LOD (µM)
Repeatability (%)
throughput (h-1)
Ref.
Enzymatic methods Amperometry
0.2-3.4 mM
40
2.4
-
[31]
Chemiluminescence
0.1-3.0
670
7.1
12
[32]
21
mM 0.01-0.1 mM
5.9
-
45
[33]
1.3-10 mM
500
-
45
[33]
9.0-130 µM
3
1
23
[34]
SI-LOV-spectrophotometry
FI-spectrophotometry
Non-enzymatic methods 0.02-1.0 mM
2.05
-
-
[14]
2.0-80 mM
19.6
-
-
[14]
5-35000 µM
1.6
3.9
45-610 µM
28
1.9
26
[34]
100-4500 µM
80
1.0
40
[34]
50-500 µM
30
1.5
22
[34]
30-615 µM
22
1
26
34]
Spectrofluorimetry
0.7-10 mM
350
-
54
[17]
Spectrophotometry
9.0-300 µM
3
-
12
[35]
20-20000 µM
5
3.8
720
This work
Amperometry
[15]
FI-spectrophotometry
CDs nanoprobemicrofluorospectrometry a
Abbreviations: FI, flow injection; LOV, lab-on-valve; SI, sequential injection.
22
Table 2: Comparison of the main characteristics of several synthesis procedures for CDs.
Synthetic approach
Microwave treatment
Ultrasonic treatment
Hydrotherm al method
Precursors
CD s size (n m)
Time for synthes is
Temperat ure for synthesis (oC)
Dialys is
Upconversi on properties
Citric acid/EDA
5
2 min
-
YES
NO
Sucrose/H2SO4
3-6
18 min
-
YES
-
Glucose/PEG
5
2 min
-
YES
-
[39]
Glycerine/PEG/se rine
3-4
10 min
-
YES
NO
[40]
Formamide
215
30 min
-
NO
NO
Ionic liquids
2-6
12 min
-
YES
NO
Glucose/NH4OH
10
24 h
Room temperatur e
YES
YES
Active carbon/H2O2
510
15 h
Room temperatur e
NO
YES
Fructose/PEG/Na OH
2.5
1 min
Room temperatur e
NO
-
BSA/H2SO4
1-2
2h
50
YES
-
EG/H2SO4
1-4
8h
140
NO
NO
Glycerol/PEGNH2
5.5
1h
230
YES
NO
Pepper
2-7
5h
180
YES
YES
Folic acid/EG
4.5
12 h
180
YES
NO
Pomelo peel
2-4
51 h
200
NO
-
[6]
Watermelon peel
2
2.5 h
220
YES
NO
[7]
23
Ref.
[37] [38]
[41] [42]
[43]
[44]
[11]
[45] [46] [47] [9] [48]
Photochemi cal treatment
Banana peel
5.5
3h
200
NO
-
[8]
Bamboo leave
3.6
6h
200
NO
NO
[10]
1 min
Room temperatu re
YES
Thi s wor k
sucrose/PEG/Na OH
8
NO
BSA, bovine serum albumin; EDA, ethylenediamine; EG, ethylene glycol; PVP, polyvinylpyrrolidone.
Highlights · · ·
Photochemical synthesis of fluorescent CDs is described for the first time. Integration of CDs synthesis and sensing within a single step is achieved. Turn on (H2O2) and turn on-off (antioxidants) fluorescent nanoprobes are obtained.
24
*Graphical Abstract (for review)
Figure
Figure
Figure
Figure
Figure
Figure
Figure