Talanta 132 (2015) 845–850
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Microwave-assisted synthesis of carbon dots and its potential as analysis of four heterocyclic aromatic amines C. López a,b, M. Zougagh b,c, M. Algarra d,n, E. Rodríguez-Castellón d, B.B. Campos e, J.C.G. Esteves da Silva e, J. Jiménez-Jiménez d, A. Ríos a,b a
Department of Analytical Chemistry and Food Technology, University of Castilla-La Mancha, 13004 Ciudad Real, Spain Regional Institute for Applied Chemistry Research, IRICA, 13004 Ciudad Real, Spain c Albacete Science and Technology Park, 2006 Albacete, Spain d Department of Inorganic Chemistry, University of Málaga, Campus de Teatinos s/n, 29071 Málaga, Spain e Centro de Investigação em Química, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, Porto, Portugal b
art ic l e i nf o
a b s t r a c t
Article history: Received 26 June 2014 Received in revised form 30 September 2014 Accepted 8 October 2014 Available online 31 October 2014
Fluorescent water soluble carbon nanoparticles, in short carbon dots (CDs), was synthesized from lactose by microwave assisted hydrochloric acid method. Characterized by TEM and DLS to obtain the morphology shape (average 10 nm in size), with a higher negative surface charge supported by the composition was obtained by XPS spectroscopy. The maximum of the emission was centered at 450 nm with a lifetime of 2.1 ns. Without further functionalization of the CDs a nanosensor was obtained that responded exponentially to HAAs in the 0.35–0.45 mg L 1 concentration range by fluorescence static quenching, demonstrated by the lifetime analysis of the CDs in presence of HAAs. Some amino compounds were selected as model for interferences to evaluate the selectivity of this method, showing a notorious added value, with recoveries around 98%. The accuracy of the method was in terms of RSD about 2.5%. The results suggest their promising applications in chemical sensing. & 2014 Elsevier B.V. All rights reserved.
Keywords: Carbon dots Heterocyclic aromatic amines Sensor Fluorescence quenching
1. Introduction The International Agency for Research on Cancer (IARC) regards some of the heterocyclic aromatic amines (HAAs) as possible human carcinogens such as (2-amino-3,4-dimethyl-imidazo [4,5-f]-quinoline, 2-amino-3,8-dimethyl-imidazo [4,5-f] quinoxaline and 2-amino-3,4,8trimethylimidazo [4,5-f]-quinoxaline, 2-amino-3,7,8-trimethyl-imidazo [4,5-f]-quinoxaline (class 2B) and 2-amino-3-methylimidazo [4,5-f]-quinoline (class 2A) [1–3]. Therefore, an important issue in Analytical Chemistry as the main source is the cooked food when the proteins are converted at high temperatures due to the incomplete their combustion process. Chromatographic methods are the most widely used when analyzed grilled, barbecued or fried meat and fish and urine samples, being HPLC–MS the main analytical technique used previous microextraction procedure [4–13]. HPLC with different detection systems such as UV [14–16] and fluorescence [17,18]; GC–MS [19–23] and by electrophoresis capillary (CE) [24–28], an extensive revision of all this techniques and compared can be found in literature [29]. Very few references to other techniques have been found in the literature for n
Corresponding author. Tel.: þ 34 952 131873; fax: þ34 952 132000. E-mail address: [email protected] (M. Algarra).
http://dx.doi.org/10.1016/j.talanta.2014.10.008 0039-9140/& 2014 Elsevier B.V. All rights reserved.
the analysis of these harmful metabolites, except for improvements in the processes of extraction prior to chromatographic analysis such as the used of supercritical fluids [27] and ionic liquids [30], making interesting the development of new alternatives as the sensitive luminescent techniques for their analysis is an important and interesting challenge in the analysis of HAAs [31]. Nanotechnology offers the best alternative, which combines the easiest design of nanoparticles with their cheap cost and luminescent properties, being the emerging system based in carbon dots (CDs) the best candidates to be applied as a detection system of any analyte to study due to its application in many other areas of analysis. These systems are the one of the lasts discover where heavily functionalized carbon networks are involved, which were firstly discovered by simple purification of carbon nanotubes and nowadays can be obtained by means a great variety of simple process [32–37]. This work shows the photoluminescent characteristics of CDs prepared from lactose using microwave process. The advantages of this treatment include faster preparation method, good fluorescence efficiency, reproducibility and stability of the obtained nanoparticles. Furthermore, the exploitation of the sensorial ability of these CDs gives rise to an excellent tool for the quantification, and without any further functionalization, of four different HAAs, by the quenching effect on the fluorescence of CDs.
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2. Experimental 2.1. Chemicals Lactose monohydrate (99%) and hydrochloric ( 37%) were purchased from Panreac S.A.U. (Barcelona, Spain), 2-amino-3,4-dimethyl-imidazo [4,5-f]-quinoline (MeIQ), 2-amino-3,8-dimethyl-imidazo [4,5-f]-quinoxaline(8-MeIQx), 2-amino-3,4,8-trimethylimidazo [4,5-f]-quinoxaline (4,8-DiMeIQx), 2-amino-3,7,8-trimethyl-imidazo [4,5-f]-quinoxaline (7,8-DiMeIQx), 2-Amino-1-methyl-6-phenyilimidazo [4,5-b]-pyridine (PhIP) and 2-amino-1-methyl-imidazo [4,5-f]quinoline (iso-IQ) (Fig. 1), were supplied from Toronto Research Chemicals Inc. (North York, ON, Canada). Ethylamine, trimethylamine, lysine, arginine and tyrosine were purchased from SigmaAldrich. Ultrapure water used throughout all experiments was purified through a Millipore system. All reagents were used as received without further purification. Fig. 1. Chemical structures of the heterocyclic aromatic amines: 2-amino-3,4dimethyl-imidazo (MeIQ); 2-amino-3,8-dimethyl-imidazo [4,5-f]-quinoxaline (8MeIQx); 2-amino-3,4,8-trimethylimidazo [4,5-f]-quinoxaline (4,8-DiMeIQx) and 2-amino-3,7,8-trimethyl-imidazo [4,5-f]-quinoxaline (7,8-DiMeIQx).
2.2. Synthesis of CDs With ultrasounds, CDs were prepared by mixing a solution of lactose 1 M (50 mL) with concentrated HCl (50 mL). The mixture
Fig. 2. (A and B) Typical TEM images of CDs thus synthesized at different magnification by assisted microwave procedure at 160 1C, and (C) The corresponding CDs size distribution histogram.
C. López et al. / Talanta 132 (2015) 845–850
was transferred into a 100 mL Teflon vessel and submitted to a hydrothermal microwave treatment at 160 1C for 15 min (ANTON Microwave 3000 at 20 bar and 750 W), obtaining a brown colour product. The sample was cooled down naturally and the resulted solution was centrifuged, filtered, neutralized to pH 7, centrifuged again and lyophilized to afford the fluorescent CDs for further characterization and use. 2.3. Characterization of CDs and data analysis PhotonTechnology International (PTI) Inc. Quanta Master 40 spectrofluorometer that was equipped with a 75 W continuous Xenon arc lamp. An ASOC-10 USB interface FeliX GX software was used for fluorescence data acquisition and also controlled the hardware for all system configurations. The slits width for excitation and emission widths were both 2 nm. All optical measurements were performed in 10 mm quartz cell at room temperature. Fluorescence lifetime analysis was done using an Edinburgh Instruments FLS920, equipped with a Xe lamp (450 W) as excitation source for steady state fluorescence measurements and monochromatic LEDs (PicoQuant PLS), controlled by a PDL 880-B system. Fluorescence decays were interpreted in τ terms of a multi-exponential: I(t)¼Aþ ΣBiexp t/ i, where A and Bi are the pre-exponential factors and τi the decay times. Transmission Electron Microscopy (TEM) analysis was carried out with a Philips CM-200. The size and zeta potential (ζ) of CDs were determined using a Zetasizer Nano ZS (Malvern Instruments, U.K.) equipped with a 4 mW HeNe laser operating at λ ¼633 nm. Size measurements were recorded with dynamic light scattering (DLS), at 25 1C in a polystyrene cell (ZEN0040) at a scattering angle of 1731 and were average of three tests. The ζ measurements were also performed at 25 1C in polycarbonate folded capillary cells, incorporated with gold plated electrodes (DTS1061) and deionized H2O was the dispersion medium. Both, size and ζ were automatically obtained by the software, using the Stokes–Einstein and the Henry equation, with the Smoluchowski approximation. X-ray photoelectron spectroscopic (XPS) studies were performed on a Physical Electronic PHI 5700 spectrometer using nonmonochromatic Mg-Ka radiation (300 W, 15 kV, 1253.6 eV) for analyzing the core-level signals of the elements of interest with a hemispherical multichannel detector. The spectra of powdered samples were recorded with a constant pass energy value at 29.35 eV, using a 720 μm diameter circular analysis area. The X-ray photoelectron spectra obtained were analyzed using PHI ACESS ESCA-V6.0F software and processed using Multipak 8.2B package. The binding energy values were referenced to adventitious carbon C 1s signal (284.8 eV). Shirley-type background and Gauss–Lorentz curves were used to determine the binding energy.
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around 10.9973.97 nm (Fig. 2C). The zeta potential (ζ) of the CDs synthesized by this procedure afforded high negative charge ( 29.171.46 mV), in accordance with previous results when carbohydrates are used as CDs precursors, where a high density of susceptible functional groups to be in the surface [35], the XPS analysis supported this hypothesis due to synthesis procedure carried out. The surface composition and elemental analysis for the resultant CDs were characterized by XPS technique. The XPS results indicate that these CDs are composed, in atomic concentration %, of C: 79.99%; O: 14.06%, Na: 3.59; Cl: 2.36%. The C 1s spectrum of CDs (Fig. 3A) is
Fig. 3. (A) C 1s and (B) O 1s cores level spectra for CDs.
2.4. Procedure For the evaluation of HAAs as possible quenchers of the CDs fluorescence in solution, microliters of stock solutions (1000 mg L 1) of chosen analytes were added to 50 μL of CDs keeping constant the final volume to 5 mL. The concentrations ranging upward from 0.5 to 10 mg L 1 were measured to obtain the Stern–Volmer plot.
3. Results and discussion An easiest, rapid and hydrothermal procedure, assisted by microwave treatment, previous purification process, was chosen to obtain CDs from lactose which exhibited a strong emission. The most attractive feature of this method is that the surface passivation step is not necessary, since the starting material. Fig. 2A and B shows the TEM images of CDs at different magnifications revealing, that the synthesis procedure without any modification, could easily disperse in water with a narrow size distribution showing a mean diameter
Fig. 4. Fluorescence spectra of CDs obtained from lactose after microwave treatment (λexc ¼350 nm; slit widths: 5.5 nm). Inset: the influence of pH on the intensity of CDs at 450 nm.
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composed of four contributions at 284.8 (81%), 286.3 (7%), 288.2 (6%) and 290.1 eV (6%). The first and main contribution at 284.8 eV is assigned to graphitic carbon plus adventitious carbon. The second one at 286.3 eV to carbonyl groups and the contributions at higher binding energy (288.2 and 290.1 eV) to carboxylic and carbonate groups, respectively. The O 1s core level spectrum shows and asymmetric signal that can be decomposed in two contribution at 531.3 eV (85%) and 532.9 eV (15%) assigned to oxygen of carbonyl and carboxylate groups (Fig. 3B). The Cl 2p3/2 signal appears at 199.1 eV, typically of chlorine covalently bonded to carbon (Cl–C). Finally, the Na 1s signal is symmetric and centred at 1071.2 eV, being assigned to Na þ as counter ion of the carboxylic group. To further elucidate the surface features of the CDs, various spectroscopic analyses were used, the ATR spectra two prominent bands are observed at 3240 and 1644 cm 1, which correspond to the stretching of –OH and C¼ O bonds, respectively. The Raman spectrum of the as-prepared CDs was also measured which, in addition to the IR features, revealed distinct peaks: (i) at 1610 cm 1 due to the carbonyl groups attached to the CDs, which are bonded to
donor groups (ii) a weak band at 1345 cm 1 typical of carbon nanostructures in this case due to the CDs core vibrations, i.e.: carbonaceous structure and (iii) at 1078 cm 1 likely arising from the C–C and C–O bending modes. Both together, the IR and Raman spectra provide spectroscopic evidences of the composition of the CD dots complementary to that of XPS in this study (Supplementary material). The influence of the thermal effect was studied over the CDs, and was observed that after the synthesis of CDs by the proposed method, when heated until 100 1C, only an increasing on the fluorescence intensity was observed, around 13% (Supplementary material). Fig. 4 shows the fluorescence spectra of CDs with a maximum of emission at 450 nm (λexc ¼350 nm), showing a full width at half maximum (FWHM) of 152 nm, relative high value, indicating their great size distribution dependence. The stability of CDs was investigated under three different conditions sunlight exposure, ambient light and dark. For this purpose, aliquots of 100-fold dilutions of CDs were prepared and exposed under these conditions during several intervals of times. In all cases the fluorescence intensity at 450 nm were recorded. In general, CDs remain stable for long periods (three months). In addition, CDs are photo-stable when they remain under continuous irradiation. No abnormalities in their fluorescence because of sunlight exposition were observed either. The influence of pH on the fluorescence activity of CDs was also studied. Inset Fig. 4 shows the pH-response of photoluminescence behavior of the different water-soluble CDs, in which the fluorescence generally tends to increase when increasing the pH of the medium in the range between 1 and 5, and decrease when the pH is high tan 5. It is noticeable than this synthesis procedure increases the fluorescence intensity when increases the pH until 5. No aggregation neither variation in the maximum emission wavelength was observed with the pH 5 for CDs. The analysis of the fluorescence decays shows that the lifetimes, calculated with a three component decay time model was: IðtÞ ¼ 1:501 102 þ 1:057 103 eð t=τ1 Þ þ 3:019 102 et=τ2 ðχ ¼ 1:155Þ
Fig. 5. Influence of HAAs on the fluorescence intensity of synthesized CDs when excited at 350 nm. (Inset the fluorescence of the four HAAs studied).
Fig. 6. Influence of the pH on the quenching effect of HAAs at 450 nm.
with τ1 ¼ 2.41 and τ2 ¼ 7.84 ns, respectively, showing a higher goodness of the fitting (χ). Luminescence decays showed that CDs produced by assisted microwave HCl treatment method have the presence of different emissive sites, at 450 nm of emission (λexc ¼ 350 nm), as supported by the presence of different functional groups supported by XPS analysis [38]. The dilution of CDs effects on the fluorescence intensity. Five dilutions of CDs in proportions 1:10, 1:20, 1:50, 1:100 and 1:200 with ultra-pure water and PBS solution were investigated. In both cases, optimized dilutions of 100-fold for CDs were observed and considered as stock solutions for their higher fluorescence intensity. The study of the interaction of different chemical species with the surface of CDs is widely reported in the literature [33–35]. It has revealed that the luminescence properties of these nanomaterials strongly depend on their surface atoms and their environment [39,40]. This work is focused in the interaction established between the different surface functional groups of the synthesized CDs with four HAAs (MeIQ, 8-MeIQx, 4,8-DiMeIQx and 2,7,8-DiMeIQx), which affect on the fluorescence intensity of the CDs (Fig. 5). For understanding the possible influence of these HAAs on the luminescence intensity of
Table 1 Interferences of other related amines on the determination of the selected HAAs, expressed in recoveries (%). Interferent
Tolerance ratio (m m 1)
MeIQ Recovery (%)
8-MeIQx Recovery (%)
4,8-DiMeIQx Recovery (%)
7,8-DiMeIQx Recovery (%)
Aliphatic amines: ethylamine and trimethylamine Aminoacids: lysine, arginine and tyrosine Others heterocyclic amines: PhIP and Iso-IQ
1000 900 250
100 95–99 100
100 98–101 99, 102
100 97–99 94, 97
100 95–97 96, 98
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CDs, the fluorescence emission spectrum of each HAAs (λex ¼350 nm) was observed. Any significant emission was observed at the 400– 650 nm range (inset in Fig. 5). When each HAAs was added to the CDs aqueous solution, a quenching effect on the CDs emission band (λem ¼450 nm) was observed. Previously to the determination of the selected HAAs, were carried out the pH study, MeIQ, 8-MeIQx, 4,8DiMeIQx and 7,8-DiMeIQx, with a pKa 5.95, 5.86, 5.56 and 5.65, respectively, have their N atom in the 3 position of imidazole ring as their most basic centre [41]. These analytes, protonated at pHo5, interact with a negative charged surface of CDs by hydrogen bonded of amine groups of HAAs and carboxylic groups of CDs, causing maximum quenching of fluorescence at pH 5 (Fig. 6). At pH45 a quenching decrease since the negative charged amino groups of HAAs. In order to assess the feasibility of the proposed analytical method, the effect of other group of amines on the fluorescence signal of CDs was also checked. This study included aliphatic amines (ethylamine and trimethylamine), aminoacids (lysine, arginine and tyrosine), and others HAAs such as (2-Amino-1-methyl-6-phenyilimidazo[4,5-b]pyridine (PhIP) and 2-amino-1-methyl-imidazo [4,5-f]-quinoline (isoIQ), that were added over the CDs ten levels of concentrations (10, 50, 100, 250, 400, 500, 600, 800, 900 and 1000 mg L 1). The results obtained indicated that these amines did not produce any noticeable effect in the fluorescence signal of the CDs with 1 mg L 1 of HAAs. The results of including significant levels of possible interferents are presented in Table 1. The results indicated that the aliphatic amines and amino acids did not quench CDs fluorescence emissions with each HAAs which in turn would give rise to erroneous HAAs levels being determined (tolerance ratio of 1000 m m 1). In the case of others HAAs such as PhIP and isoIQ, the tolerance ratio was 250. The dependence of the measured luminescence intensity signal with increasing concentrations of HAAs was observed to follow the well-known Stern–Volmer equation (I0/I ¼1 þ Ksv [Q]), where I0 is the luminescence signal at 450 nm of CDs in absence of HAAs and I is the same signal in the presence of increasing concentration of HAAs. Hence, (I0/I) were plotted versus each HAA concentration. It is interesting to remark that this plot did not fit a conventional linear equation. An upward
Fig. 7. Calibration graphs obtained for HAAs at 450 nm.
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curvature was observed, typical behavior of charged quenchers Fig. 7 shows the logarithmic known Stern–Volmer plots and calibration equations of MeIQ, 8-MeIQx, 4,8-DiMeIQx and 7,8DiMeIQx. These molecules do not contact at all in the surface of the CDs, only a partial amount of molecules of HAAs, indicating that both dynamic and static quenching seem to act together, here suggesting a more complex quenching model [42,43]. However, when ln(I0/I) was plotted versus the concentration of each HAAs a good linear relationship (r2 4 0.995) was obtained, in other words the quenching of the fluorescence of CDs has an exponential α relationship with the concentration of HAAs (I0/I ¼e [HAAs]), where α represents the constant associated to the quenching effect. Therefore, this signal could be used to develop a HAAs determination method based on luminescence quenching of CDs by DiMeIQx at optimum conditions. The precision in terms of relative standard deviation (RSD) was 0.25, 0.35, 0.57 and 0.75% for MeIQ, 8-MeIQx, 4,8-DiMeIQx and 7,8-DiMeIQx, respectively; being the reproducibility (for 1 mg L 1 of each HAA, n¼5) of RSD value less than of 2.5% in all cases. The limit of quantification, were 0.35, 0.41, 0.45 and 0.36 mg L 1 for MeIQ, 8-MeIQx, 4,8-DiMeIQx and 7,8-DiMeIQx, respectively. To check this effect, the analysis of the fluorescence decays shows that the lifetimes of the components (Table 2), calculated with a component decay time model for CDs, in the presence of increasing concentration of 4,8-DiMeIQx is not particularly sensitive to the presence of the quencher. They do not depend of the HAA concentration, demonstrating the static quenching effect over CDs linked to the formation of a stable complex between the fluorophore and 4,8DiMeIQx in this case. Fig. 8 shows the lifetime decay curves of the fluorescence emission, which can be fitted to four components. 4. Conclusions In summary, a fluorescent analytical method based on the quenching of the un-functionalized carbon fluorescent nanoparticles,
Fig. 8. Fluorescence lifetime curves of CDs obtained from lactose. A four component exponential-decay curve is fitted to the selected data after background subtraction. Inset is the magnification of the bottom of the decay curves at different concentrations.
Table 2 Lifetime intensity decays of CDs obtained from lactose treated with HCl. Fluorescence Lifetime Parameters
HAA ¼ 0 (mg L 1)
HAA ¼ 8 (mg L 1)
HAA ¼ 14 (mg L 1)
τI(ns)/Bi (10 2) τI(ns)/Bi (10 2) τI(ns)/Bi (10 2) τI(ns)/Bi (10 2) χ
0.28 7 0.03/2.40 7 0.05 1.107 0.20/2.32 7 0.02 2.93 7 0.07/0.1607 0.003 7.90 7 0.20/6.83 7 0.11 1.034 6.077 0.30
0.217 0.05/3.94 7 0.07 0.767 0.08/2.00 70.02 2.69 7 0.09/0.33970.004 6.40 7 0.20/7.30 7 0.18 1.091 13.497 0.35
0.25 7 0.07/7.92 7 0.13 0.93 7 0.08/3.25 7 0.06 2.75 7 0.04/0.238 7 0.003 7.30 7 0.40/1.91 70.02 1.120 10.78 7 0.31
A
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obtained by the assisted microwave treatment of lactose by HCl, and was used as potential system to quantify heterocyclic aromatic amines at μM range of concentration static quenching effect was demonstrated, after analysis of the fluorescence lifetime decay curves, no effect was detected when used 4,8-DiMeIQx as heterocyclic aromatic amine probe at different concentration. CDs can selectively recognize certain homologues amines, and give a sensitive signal response, showing their promising use in the chemical sensing HAAs. Acknowledgements Authors are gratefully to Grant SFRH/BD/84318/2012 to FCT (Lisbon, Portugal) and Andalucía Tech Program from Univ Málaga (Spain). Financial support from the Spanish Ministry of Economy and Competitiveness (CTQ2013-48411-P) is gratefully acknowledged. The support given through an “INCRECYT” research contract to M. Zougagh is also acknowledged. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2014.10.008. References [1] IARC, Monographs on the Evaluation of Carcinogenic Risks to Humans. Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins, vol. 56, 1993. [2] K. John, S. Beedanagari, Heterocyclic Aromatic Amines , Encyclopaedia of Toxicology (2014) 855–863 (third ed.). [3] K. Puangsombat, P. Gadgil, T.A. Houser, M.C. Hunt, J.S. Smith, Meat Sci. 90 (2012) 739. [4] P. Pais, E. Moyano, L. Puignou, M.T. Galceran., J. Chromatogr. A 778 (1997) 207. [5] E. Barceló-Barrachina, E. Moyano, M.T. Galcerán, J. Chromatogr. A 1054 (2004) 409. [6] M.R. Khan, R. Busquets, F.J. Santos, L.J. Puignou, J. Chromatogr. A 1194 (2008) 155. [7] T. Polak, D. Došler, B. Žlender, L. Gašperlin, LWT Food Sci. Technol 42 (2009) 504. [8] M. Iwasaki, H. Kataoka, J. Ishihara, R. Takachi, G.S. Hamada, S. Sharma, L. Le Marchand, S. Tsugane, J. Food Compos. Anal. 23 (2010) 61. [9] H. Kataoka, T. Inoue, K. Saito, H. Kato, K. Masuda, Anal. Chim. Acta 786 (2013) 54–60. [10] J.H. Shin, Y.C. Na, J.H. Chung, S. Gorinstein, Y.G. Ahn, Y. Gyong, Anal. Bioanal. Chem 447 (2014) 169.
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Talanta journal homepage: www.elsevier.com/locate/talanta
Microwave-assisted synthesis of carbon dots and its potential as analysis of four heterocyclic aromatic amines C. López a,b, M. Zougagh b,c, M. Algarra d,n, E. Rodríguez-Castellón d, B.B. Campos e, J.C.G. Esteves da Silva e, J. Jiménez-Jiménez d, A. Ríos a,b a
Department of Analytical Chemistry and Food Technology, University of Castilla-La Mancha, 13004 Ciudad Real, Spain Regional Institute for Applied Chemistry Research, IRICA, 13004 Ciudad Real, Spain c Albacete Science and Technology Park, 2006 Albacete, Spain d Department of Inorganic Chemistry, University of Málaga, Campus de Teatinos s/n, 29071 Málaga, Spain e Centro de Investigação em Química, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, Porto, Portugal b
art ic l e i nf o
a b s t r a c t
Article history: Received 26 June 2014 Received in revised form 30 September 2014 Accepted 8 October 2014 Available online 31 October 2014
Fluorescent water soluble carbon nanoparticles, in short carbon dots (CDs), was synthesized from lactose by microwave assisted hydrochloric acid method. Characterized by TEM and DLS to obtain the morphology shape (average 10 nm in size), with a higher negative surface charge supported by the composition was obtained by XPS spectroscopy. The maximum of the emission was centered at 450 nm with a lifetime of 2.1 ns. Without further functionalization of the CDs a nanosensor was obtained that responded exponentially to HAAs in the 0.35–0.45 mg L 1 concentration range by fluorescence static quenching, demonstrated by the lifetime analysis of the CDs in presence of HAAs. Some amino compounds were selected as model for interferences to evaluate the selectivity of this method, showing a notorious added value, with recoveries around 98%. The accuracy of the method was in terms of RSD about 2.5%. The results suggest their promising applications in chemical sensing. & 2014 Elsevier B.V. All rights reserved.
Keywords: Carbon dots Heterocyclic aromatic amines Sensor Fluorescence quenching
1. Introduction The International Agency for Research on Cancer (IARC) regards some of the heterocyclic aromatic amines (HAAs) as possible human carcinogens such as (2-amino-3,4-dimethyl-imidazo [4,5-f]-quinoline, 2-amino-3,8-dimethyl-imidazo [4,5-f] quinoxaline and 2-amino-3,4,8trimethylimidazo [4,5-f]-quinoxaline, 2-amino-3,7,8-trimethyl-imidazo [4,5-f]-quinoxaline (class 2B) and 2-amino-3-methylimidazo [4,5-f]-quinoline (class 2A) [1–3]. Therefore, an important issue in Analytical Chemistry as the main source is the cooked food when the proteins are converted at high temperatures due to the incomplete their combustion process. Chromatographic methods are the most widely used when analyzed grilled, barbecued or fried meat and fish and urine samples, being HPLC–MS the main analytical technique used previous microextraction procedure [4–13]. HPLC with different detection systems such as UV [14–16] and fluorescence [17,18]; GC–MS [19–23] and by electrophoresis capillary (CE) [24–28], an extensive revision of all this techniques and compared can be found in literature [29]. Very few references to other techniques have been found in the literature for n
Corresponding author. Tel.: þ 34 952 131873; fax: þ34 952 132000. E-mail address: [email protected] (M. Algarra).
http://dx.doi.org/10.1016/j.talanta.2014.10.008 0039-9140/& 2014 Elsevier B.V. All rights reserved.
the analysis of these harmful metabolites, except for improvements in the processes of extraction prior to chromatographic analysis such as the used of supercritical fluids [27] and ionic liquids [30], making interesting the development of new alternatives as the sensitive luminescent techniques for their analysis is an important and interesting challenge in the analysis of HAAs [31]. Nanotechnology offers the best alternative, which combines the easiest design of nanoparticles with their cheap cost and luminescent properties, being the emerging system based in carbon dots (CDs) the best candidates to be applied as a detection system of any analyte to study due to its application in many other areas of analysis. These systems are the one of the lasts discover where heavily functionalized carbon networks are involved, which were firstly discovered by simple purification of carbon nanotubes and nowadays can be obtained by means a great variety of simple process [32–37]. This work shows the photoluminescent characteristics of CDs prepared from lactose using microwave process. The advantages of this treatment include faster preparation method, good fluorescence efficiency, reproducibility and stability of the obtained nanoparticles. Furthermore, the exploitation of the sensorial ability of these CDs gives rise to an excellent tool for the quantification, and without any further functionalization, of four different HAAs, by the quenching effect on the fluorescence of CDs.
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2. Experimental 2.1. Chemicals Lactose monohydrate (99%) and hydrochloric ( 37%) were purchased from Panreac S.A.U. (Barcelona, Spain), 2-amino-3,4-dimethyl-imidazo [4,5-f]-quinoline (MeIQ), 2-amino-3,8-dimethyl-imidazo [4,5-f]-quinoxaline(8-MeIQx), 2-amino-3,4,8-trimethylimidazo [4,5-f]-quinoxaline (4,8-DiMeIQx), 2-amino-3,7,8-trimethyl-imidazo [4,5-f]-quinoxaline (7,8-DiMeIQx), 2-Amino-1-methyl-6-phenyilimidazo [4,5-b]-pyridine (PhIP) and 2-amino-1-methyl-imidazo [4,5-f]quinoline (iso-IQ) (Fig. 1), were supplied from Toronto Research Chemicals Inc. (North York, ON, Canada). Ethylamine, trimethylamine, lysine, arginine and tyrosine were purchased from SigmaAldrich. Ultrapure water used throughout all experiments was purified through a Millipore system. All reagents were used as received without further purification. Fig. 1. Chemical structures of the heterocyclic aromatic amines: 2-amino-3,4dimethyl-imidazo (MeIQ); 2-amino-3,8-dimethyl-imidazo [4,5-f]-quinoxaline (8MeIQx); 2-amino-3,4,8-trimethylimidazo [4,5-f]-quinoxaline (4,8-DiMeIQx) and 2-amino-3,7,8-trimethyl-imidazo [4,5-f]-quinoxaline (7,8-DiMeIQx).
2.2. Synthesis of CDs With ultrasounds, CDs were prepared by mixing a solution of lactose 1 M (50 mL) with concentrated HCl (50 mL). The mixture
Fig. 2. (A and B) Typical TEM images of CDs thus synthesized at different magnification by assisted microwave procedure at 160 1C, and (C) The corresponding CDs size distribution histogram.
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was transferred into a 100 mL Teflon vessel and submitted to a hydrothermal microwave treatment at 160 1C for 15 min (ANTON Microwave 3000 at 20 bar and 750 W), obtaining a brown colour product. The sample was cooled down naturally and the resulted solution was centrifuged, filtered, neutralized to pH 7, centrifuged again and lyophilized to afford the fluorescent CDs for further characterization and use. 2.3. Characterization of CDs and data analysis PhotonTechnology International (PTI) Inc. Quanta Master 40 spectrofluorometer that was equipped with a 75 W continuous Xenon arc lamp. An ASOC-10 USB interface FeliX GX software was used for fluorescence data acquisition and also controlled the hardware for all system configurations. The slits width for excitation and emission widths were both 2 nm. All optical measurements were performed in 10 mm quartz cell at room temperature. Fluorescence lifetime analysis was done using an Edinburgh Instruments FLS920, equipped with a Xe lamp (450 W) as excitation source for steady state fluorescence measurements and monochromatic LEDs (PicoQuant PLS), controlled by a PDL 880-B system. Fluorescence decays were interpreted in τ terms of a multi-exponential: I(t)¼Aþ ΣBiexp t/ i, where A and Bi are the pre-exponential factors and τi the decay times. Transmission Electron Microscopy (TEM) analysis was carried out with a Philips CM-200. The size and zeta potential (ζ) of CDs were determined using a Zetasizer Nano ZS (Malvern Instruments, U.K.) equipped with a 4 mW HeNe laser operating at λ ¼633 nm. Size measurements were recorded with dynamic light scattering (DLS), at 25 1C in a polystyrene cell (ZEN0040) at a scattering angle of 1731 and were average of three tests. The ζ measurements were also performed at 25 1C in polycarbonate folded capillary cells, incorporated with gold plated electrodes (DTS1061) and deionized H2O was the dispersion medium. Both, size and ζ were automatically obtained by the software, using the Stokes–Einstein and the Henry equation, with the Smoluchowski approximation. X-ray photoelectron spectroscopic (XPS) studies were performed on a Physical Electronic PHI 5700 spectrometer using nonmonochromatic Mg-Ka radiation (300 W, 15 kV, 1253.6 eV) for analyzing the core-level signals of the elements of interest with a hemispherical multichannel detector. The spectra of powdered samples were recorded with a constant pass energy value at 29.35 eV, using a 720 μm diameter circular analysis area. The X-ray photoelectron spectra obtained were analyzed using PHI ACESS ESCA-V6.0F software and processed using Multipak 8.2B package. The binding energy values were referenced to adventitious carbon C 1s signal (284.8 eV). Shirley-type background and Gauss–Lorentz curves were used to determine the binding energy.
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around 10.9973.97 nm (Fig. 2C). The zeta potential (ζ) of the CDs synthesized by this procedure afforded high negative charge ( 29.171.46 mV), in accordance with previous results when carbohydrates are used as CDs precursors, where a high density of susceptible functional groups to be in the surface [35], the XPS analysis supported this hypothesis due to synthesis procedure carried out. The surface composition and elemental analysis for the resultant CDs were characterized by XPS technique. The XPS results indicate that these CDs are composed, in atomic concentration %, of C: 79.99%; O: 14.06%, Na: 3.59; Cl: 2.36%. The C 1s spectrum of CDs (Fig. 3A) is
Fig. 3. (A) C 1s and (B) O 1s cores level spectra for CDs.
2.4. Procedure For the evaluation of HAAs as possible quenchers of the CDs fluorescence in solution, microliters of stock solutions (1000 mg L 1) of chosen analytes were added to 50 μL of CDs keeping constant the final volume to 5 mL. The concentrations ranging upward from 0.5 to 10 mg L 1 were measured to obtain the Stern–Volmer plot.
3. Results and discussion An easiest, rapid and hydrothermal procedure, assisted by microwave treatment, previous purification process, was chosen to obtain CDs from lactose which exhibited a strong emission. The most attractive feature of this method is that the surface passivation step is not necessary, since the starting material. Fig. 2A and B shows the TEM images of CDs at different magnifications revealing, that the synthesis procedure without any modification, could easily disperse in water with a narrow size distribution showing a mean diameter
Fig. 4. Fluorescence spectra of CDs obtained from lactose after microwave treatment (λexc ¼350 nm; slit widths: 5.5 nm). Inset: the influence of pH on the intensity of CDs at 450 nm.
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composed of four contributions at 284.8 (81%), 286.3 (7%), 288.2 (6%) and 290.1 eV (6%). The first and main contribution at 284.8 eV is assigned to graphitic carbon plus adventitious carbon. The second one at 286.3 eV to carbonyl groups and the contributions at higher binding energy (288.2 and 290.1 eV) to carboxylic and carbonate groups, respectively. The O 1s core level spectrum shows and asymmetric signal that can be decomposed in two contribution at 531.3 eV (85%) and 532.9 eV (15%) assigned to oxygen of carbonyl and carboxylate groups (Fig. 3B). The Cl 2p3/2 signal appears at 199.1 eV, typically of chlorine covalently bonded to carbon (Cl–C). Finally, the Na 1s signal is symmetric and centred at 1071.2 eV, being assigned to Na þ as counter ion of the carboxylic group. To further elucidate the surface features of the CDs, various spectroscopic analyses were used, the ATR spectra two prominent bands are observed at 3240 and 1644 cm 1, which correspond to the stretching of –OH and C¼ O bonds, respectively. The Raman spectrum of the as-prepared CDs was also measured which, in addition to the IR features, revealed distinct peaks: (i) at 1610 cm 1 due to the carbonyl groups attached to the CDs, which are bonded to
donor groups (ii) a weak band at 1345 cm 1 typical of carbon nanostructures in this case due to the CDs core vibrations, i.e.: carbonaceous structure and (iii) at 1078 cm 1 likely arising from the C–C and C–O bending modes. Both together, the IR and Raman spectra provide spectroscopic evidences of the composition of the CD dots complementary to that of XPS in this study (Supplementary material). The influence of the thermal effect was studied over the CDs, and was observed that after the synthesis of CDs by the proposed method, when heated until 100 1C, only an increasing on the fluorescence intensity was observed, around 13% (Supplementary material). Fig. 4 shows the fluorescence spectra of CDs with a maximum of emission at 450 nm (λexc ¼350 nm), showing a full width at half maximum (FWHM) of 152 nm, relative high value, indicating their great size distribution dependence. The stability of CDs was investigated under three different conditions sunlight exposure, ambient light and dark. For this purpose, aliquots of 100-fold dilutions of CDs were prepared and exposed under these conditions during several intervals of times. In all cases the fluorescence intensity at 450 nm were recorded. In general, CDs remain stable for long periods (three months). In addition, CDs are photo-stable when they remain under continuous irradiation. No abnormalities in their fluorescence because of sunlight exposition were observed either. The influence of pH on the fluorescence activity of CDs was also studied. Inset Fig. 4 shows the pH-response of photoluminescence behavior of the different water-soluble CDs, in which the fluorescence generally tends to increase when increasing the pH of the medium in the range between 1 and 5, and decrease when the pH is high tan 5. It is noticeable than this synthesis procedure increases the fluorescence intensity when increases the pH until 5. No aggregation neither variation in the maximum emission wavelength was observed with the pH 5 for CDs. The analysis of the fluorescence decays shows that the lifetimes, calculated with a three component decay time model was: IðtÞ ¼ 1:501 102 þ 1:057 103 eð t=τ1 Þ þ 3:019 102 et=τ2 ðχ ¼ 1:155Þ
Fig. 5. Influence of HAAs on the fluorescence intensity of synthesized CDs when excited at 350 nm. (Inset the fluorescence of the four HAAs studied).
Fig. 6. Influence of the pH on the quenching effect of HAAs at 450 nm.
with τ1 ¼ 2.41 and τ2 ¼ 7.84 ns, respectively, showing a higher goodness of the fitting (χ). Luminescence decays showed that CDs produced by assisted microwave HCl treatment method have the presence of different emissive sites, at 450 nm of emission (λexc ¼ 350 nm), as supported by the presence of different functional groups supported by XPS analysis [38]. The dilution of CDs effects on the fluorescence intensity. Five dilutions of CDs in proportions 1:10, 1:20, 1:50, 1:100 and 1:200 with ultra-pure water and PBS solution were investigated. In both cases, optimized dilutions of 100-fold for CDs were observed and considered as stock solutions for their higher fluorescence intensity. The study of the interaction of different chemical species with the surface of CDs is widely reported in the literature [33–35]. It has revealed that the luminescence properties of these nanomaterials strongly depend on their surface atoms and their environment [39,40]. This work is focused in the interaction established between the different surface functional groups of the synthesized CDs with four HAAs (MeIQ, 8-MeIQx, 4,8-DiMeIQx and 2,7,8-DiMeIQx), which affect on the fluorescence intensity of the CDs (Fig. 5). For understanding the possible influence of these HAAs on the luminescence intensity of
Table 1 Interferences of other related amines on the determination of the selected HAAs, expressed in recoveries (%). Interferent
Tolerance ratio (m m 1)
MeIQ Recovery (%)
8-MeIQx Recovery (%)
4,8-DiMeIQx Recovery (%)
7,8-DiMeIQx Recovery (%)
Aliphatic amines: ethylamine and trimethylamine Aminoacids: lysine, arginine and tyrosine Others heterocyclic amines: PhIP and Iso-IQ
1000 900 250
100 95–99 100
100 98–101 99, 102
100 97–99 94, 97
100 95–97 96, 98
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CDs, the fluorescence emission spectrum of each HAAs (λex ¼350 nm) was observed. Any significant emission was observed at the 400– 650 nm range (inset in Fig. 5). When each HAAs was added to the CDs aqueous solution, a quenching effect on the CDs emission band (λem ¼450 nm) was observed. Previously to the determination of the selected HAAs, were carried out the pH study, MeIQ, 8-MeIQx, 4,8DiMeIQx and 7,8-DiMeIQx, with a pKa 5.95, 5.86, 5.56 and 5.65, respectively, have their N atom in the 3 position of imidazole ring as their most basic centre [41]. These analytes, protonated at pHo5, interact with a negative charged surface of CDs by hydrogen bonded of amine groups of HAAs and carboxylic groups of CDs, causing maximum quenching of fluorescence at pH 5 (Fig. 6). At pH45 a quenching decrease since the negative charged amino groups of HAAs. In order to assess the feasibility of the proposed analytical method, the effect of other group of amines on the fluorescence signal of CDs was also checked. This study included aliphatic amines (ethylamine and trimethylamine), aminoacids (lysine, arginine and tyrosine), and others HAAs such as (2-Amino-1-methyl-6-phenyilimidazo[4,5-b]pyridine (PhIP) and 2-amino-1-methyl-imidazo [4,5-f]-quinoline (isoIQ), that were added over the CDs ten levels of concentrations (10, 50, 100, 250, 400, 500, 600, 800, 900 and 1000 mg L 1). The results obtained indicated that these amines did not produce any noticeable effect in the fluorescence signal of the CDs with 1 mg L 1 of HAAs. The results of including significant levels of possible interferents are presented in Table 1. The results indicated that the aliphatic amines and amino acids did not quench CDs fluorescence emissions with each HAAs which in turn would give rise to erroneous HAAs levels being determined (tolerance ratio of 1000 m m 1). In the case of others HAAs such as PhIP and isoIQ, the tolerance ratio was 250. The dependence of the measured luminescence intensity signal with increasing concentrations of HAAs was observed to follow the well-known Stern–Volmer equation (I0/I ¼1 þ Ksv [Q]), where I0 is the luminescence signal at 450 nm of CDs in absence of HAAs and I is the same signal in the presence of increasing concentration of HAAs. Hence, (I0/I) were plotted versus each HAA concentration. It is interesting to remark that this plot did not fit a conventional linear equation. An upward
Fig. 7. Calibration graphs obtained for HAAs at 450 nm.
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curvature was observed, typical behavior of charged quenchers Fig. 7 shows the logarithmic known Stern–Volmer plots and calibration equations of MeIQ, 8-MeIQx, 4,8-DiMeIQx and 7,8DiMeIQx. These molecules do not contact at all in the surface of the CDs, only a partial amount of molecules of HAAs, indicating that both dynamic and static quenching seem to act together, here suggesting a more complex quenching model [42,43]. However, when ln(I0/I) was plotted versus the concentration of each HAAs a good linear relationship (r2 4 0.995) was obtained, in other words the quenching of the fluorescence of CDs has an exponential α relationship with the concentration of HAAs (I0/I ¼e [HAAs]), where α represents the constant associated to the quenching effect. Therefore, this signal could be used to develop a HAAs determination method based on luminescence quenching of CDs by DiMeIQx at optimum conditions. The precision in terms of relative standard deviation (RSD) was 0.25, 0.35, 0.57 and 0.75% for MeIQ, 8-MeIQx, 4,8-DiMeIQx and 7,8-DiMeIQx, respectively; being the reproducibility (for 1 mg L 1 of each HAA, n¼5) of RSD value less than of 2.5% in all cases. The limit of quantification, were 0.35, 0.41, 0.45 and 0.36 mg L 1 for MeIQ, 8-MeIQx, 4,8-DiMeIQx and 7,8-DiMeIQx, respectively. To check this effect, the analysis of the fluorescence decays shows that the lifetimes of the components (Table 2), calculated with a component decay time model for CDs, in the presence of increasing concentration of 4,8-DiMeIQx is not particularly sensitive to the presence of the quencher. They do not depend of the HAA concentration, demonstrating the static quenching effect over CDs linked to the formation of a stable complex between the fluorophore and 4,8DiMeIQx in this case. Fig. 8 shows the lifetime decay curves of the fluorescence emission, which can be fitted to four components. 4. Conclusions In summary, a fluorescent analytical method based on the quenching of the un-functionalized carbon fluorescent nanoparticles,
Fig. 8. Fluorescence lifetime curves of CDs obtained from lactose. A four component exponential-decay curve is fitted to the selected data after background subtraction. Inset is the magnification of the bottom of the decay curves at different concentrations.
Table 2 Lifetime intensity decays of CDs obtained from lactose treated with HCl. Fluorescence Lifetime Parameters
HAA ¼ 0 (mg L 1)
HAA ¼ 8 (mg L 1)
HAA ¼ 14 (mg L 1)
τI(ns)/Bi (10 2) τI(ns)/Bi (10 2) τI(ns)/Bi (10 2) τI(ns)/Bi (10 2) χ
0.28 7 0.03/2.40 7 0.05 1.107 0.20/2.32 7 0.02 2.93 7 0.07/0.1607 0.003 7.90 7 0.20/6.83 7 0.11 1.034 6.077 0.30
0.217 0.05/3.94 7 0.07 0.767 0.08/2.00 70.02 2.69 7 0.09/0.33970.004 6.40 7 0.20/7.30 7 0.18 1.091 13.497 0.35
0.25 7 0.07/7.92 7 0.13 0.93 7 0.08/3.25 7 0.06 2.75 7 0.04/0.238 7 0.003 7.30 7 0.40/1.91 70.02 1.120 10.78 7 0.31
A
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obtained by the assisted microwave treatment of lactose by HCl, and was used as potential system to quantify heterocyclic aromatic amines at μM range of concentration static quenching effect was demonstrated, after analysis of the fluorescence lifetime decay curves, no effect was detected when used 4,8-DiMeIQx as heterocyclic aromatic amine probe at different concentration. CDs can selectively recognize certain homologues amines, and give a sensitive signal response, showing their promising use in the chemical sensing HAAs. Acknowledgements Authors are gratefully to Grant SFRH/BD/84318/2012 to FCT (Lisbon, Portugal) and Andalucía Tech Program from Univ Málaga (Spain). Financial support from the Spanish Ministry of Economy and Competitiveness (CTQ2013-48411-P) is gratefully acknowledged. The support given through an “INCRECYT” research contract to M. Zougagh is also acknowledged. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2014.10.008. References [1] IARC, Monographs on the Evaluation of Carcinogenic Risks to Humans. Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins, vol. 56, 1993. [2] K. John, S. Beedanagari, Heterocyclic Aromatic Amines , Encyclopaedia of Toxicology (2014) 855–863 (third ed.). [3] K. Puangsombat, P. Gadgil, T.A. Houser, M.C. Hunt, J.S. Smith, Meat Sci. 90 (2012) 739. [4] P. Pais, E. Moyano, L. Puignou, M.T. Galceran., J. Chromatogr. A 778 (1997) 207. [5] E. Barceló-Barrachina, E. Moyano, M.T. Galcerán, J. Chromatogr. A 1054 (2004) 409. [6] M.R. Khan, R. Busquets, F.J. Santos, L.J. Puignou, J. Chromatogr. A 1194 (2008) 155. [7] T. Polak, D. Došler, B. Žlender, L. Gašperlin, LWT Food Sci. Technol 42 (2009) 504. [8] M. Iwasaki, H. Kataoka, J. Ishihara, R. Takachi, G.S. Hamada, S. Sharma, L. Le Marchand, S. Tsugane, J. Food Compos. Anal. 23 (2010) 61. [9] H. Kataoka, T. Inoue, K. Saito, H. Kato, K. Masuda, Anal. Chim. Acta 786 (2013) 54–60. [10] J.H. Shin, Y.C. Na, J.H. Chung, S. Gorinstein, Y.G. Ahn, Y. Gyong, Anal. Bioanal. Chem 447 (2014) 169.
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