- Email: [email protected]
Determination of L-Cysteine Based on Energy Transfer between Cu2-xSe Nanoparticles and Rhodamine B
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 44, Issue 10, October 2016 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2016, 44(10), 1482–1486.
RESEARCH PAPER
Determination of L-Cysteine Based on Energy Transfer between Cu2-xSe Nanoparticles and Rhodamine B WANG Xue, YANG Kun-Cheng, MAO Zhi-Yuan, HUANG Cheng-Zhi*, WANG Jian* Key Laboratory on Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
Abstract: The fluorescence of Rhodamine B (RhB) could be quenched by the manner of photo-induced electron transfer with Cu2-xSe nanoparticles as the energy receptor and RhB as the energy donor. However, L-cysteine (L-Cys) was capable of recovering the fluorescence of Rhodamine B, and the recovered fluorescence intensity was proportional to the concentrations of L-Cys. Based on that, a novel method for detecting L-Cys was established. After mixing L-Cys and RhB pretreated by Cu2-xSe nanoparticles at pH 4.6 and 30 oC for 2 min, a linear relationship was obtained between the fluorescence intensity of RhB at 575 nm and the concentrations of L-Cys in the range of 2.5 × 10–7–1.1 × 10–6 M. This method was used for the determination of L-cys with a detection limit (3σ/k) of 5.5 × 10–8 M. The common amino acids presented little interference for the detection of L-Cys. Key Words: Cu2-xSe nanoparticles; L-cysteine; Photo-induced electron transfer
1
Introduction
As one common amino acid in living organisms, L-cysteine (L-Cys) is mainly found in protein and glutathione, which is associated with the physiological functions of nutrition and immunity in animals[1]. Currently, a verity of methods are developed for L-Cys detection, including electrochemistry[2], fluorescence[3,4], light scattering[5,6] and colorimetry[7–9], etc. Our group established the light scattering technique[4] and colorimetric methods[8,9] for L-Cys sensing with gold nanoparticles (AuNPs) and silver nanoparticles as optical probes. Based on the Au-S covalent bond between L-Cys and AuNPs, as well as the electrostatic interaction between COO– and NH4+ among L-Cys molecules, AuNPs aggregated with the enhanced resonance light scattering signals, which was developed for L-Cys sensing. This light scattering technique supplied a sensitive and selective result; however, it would take 25 min to complete the process[5]. Moreover, AuNPs possessed the outstanding catalytic activity, which could be inhibited
when formed aggregates induced by L-Cys, accompanied by a color change from dark blue to light blue. This finding enabled the visual detection of L-Cys with very sensitive result; however, the process took as long as 60 min[9]. Hence, it is highly desirable to develop speedy approaches to detect L-Cys. The non-stoichiometric Cu2-xSe nanoparticles (Cu2-xSe NPs) are a kind of chalcogenide semiconductor copper nanomaterials, which hold remarkable features, such as unique optical properties, adjustable size, and superior thermal properties[10]. The detection principle is presented in Scheme 1. Cu2-xSe NPs act as the energy acceptor to quench the fluorescence of energy donor-Rhodamine B (RhB). However, L-Cys is capable of recovering the quenched fluorescence of RhB. Based on that, a fluorescent method is established for detecting L-Cys. In this work, the energy transfer was mainly attributed to the photo-induced charge transfer. Wherein, RhB was easier to accept electron at excited state, and electron transfer happened between Cu2-xSe and RhB, resulting in the RhB fluorescence quenching.
________________________ Received 27 May 2016; accepted 14 August 2016 *Corresponding author. Email: [email protected]; [email protected] This work was supported by the National Natural Science Foundation of China (No. 21405123), the Fund of Chongqing Fundamental and Advanced Research Project of China (No. cstc2014jcyjA50006), the National Students' Innovative Training Project of Southwest University of China (No. 201610635050) and the College of Pharmaceutical Sciences Innovation and Entrepreneurship Project of Southwest University, China. Copyright © 2016, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(16)60960-X
WANG Xue et al. / Chinese Journal of Analytical Chemistry, 2016, 44(10): 1482–1486
then stored at 4 ºC. 2.2.2
Scheme 1 Schematic diagram of the detection of L-Cysteine with Cu2-xSe NPs
2
Fluorescence detection
Approximately 0.20 mL of RhB (3.1 × 10–6 M), 0.20 mL of Cu2-xSe NPs (2.5 × 10–7 M) and 0.10 mL of buffer (pH 4.6) were added into a 1.5-mL tube. After stirring, L-Cys at different doses was introduced. The resultant mixture was diluted to 1.0 mL with H2O, which was then fully stirred and stored at 30 ºC for 2 min. The fluorescence spectra were scanned on an F-2700 spectrophotometer at excitation wavelength of 540 nm and the slits of 5 nm.
Experimental 3
2.1
3.1 The absorption and fluorescence spectra were scanned with a UV-2600 spectrophotometer (Shimadzu, Japan) and an F-2700 spectrophotometer (Hitachi, Japan), respectively. A FL-TCSPC fluorescence spectrophotometer (Horiba Jobin Yvon Inc., France) provided the fluorescence lifetime of RhB. Both the scanning electron microscopy (SEM) imaging and transmission electron microscopy (TEM) imaging of Cu2-xSe NPs were acquired with an S-4800 scanning electron microscope (Hitachi, Japan). Zeta potential was carried out on a Nanosizer (ZEN3600, Malvern). A HCJ-6D magnetic stirrer (Changzhou, China) and a pHS-3C pH-meter (Chengdu, China) were employed to mix solutions and measure the acidity of solutions, respectively. Cu2-xSe NPs were purified with a H1650W high speed centrifuge (Hunan, China). The solutions of polyvinylpyrrolidone (PVP, Mw 55 kDa), SeO2, Vitamin c (Vc), Cu(CH3COO)2·H2O, RhB and L-Cys were stored at 4 ºC. All reagents were of analytical grade and used as received without further purification. The acidity of solutions was adjusted with citric acid-citrate sodium buffer. Doubly distilled water was used as solvent throughout this work. 2.2 2.2.1
Results and discussion
Instruments and reagents
Experimental methods Preparation of Cu2-xSe NPs
The non-stoichiometric Cu2-xSe NPs were prepared by a simple one-pot method[11]. Firstly, 1.6 mL of PVP (5 g L–1) and 5.5 mL of water were mixed in a round-bottom flask, and then 0.1 mL of SeO2 (0.1 M) and 0.3 mL of Vc (0.2 M) were introduced. After stirring for 10 min with a magnetic stirrer, a mixture of 0.1 mL of Cu(CH3COO)2·H2O (0.2 M) and 0.4 mL of Vc (0.2 M) was dropped into the flask under vigorous stirring. The resultant mixture was allowed to vigorously stir at 30 ºC for 10 h until a green solution was obtained, which was then purified by a 3500-kDa dialysis membrane for 24 h. Finally, Cu2-xSe NPs were further purified with centrifuge and
Characterization of Cu2-xSe NPs
As shown in Fig.1A and Fig.1B, the SEM and TEM images illustrated that the as-prepared Cu2-xSe NPs were well monodispersed and uniform. The size distribution of Cu2-xSe NPs was analyzed with the Nano-Measurer software. The results indicated that the diameters of Cu2-xSe NPs were mainly presented in the range of 62.4–99.2 nm with an average size of 79.2 nm (Fig.1C). 3.2
Detection of cysteine based on the fluorescence change of RhB
As shown in Fig.2A, the fluorescence of RhB at 575 nm was quenched by Cu2-xSe NPs, which was closely related to the concentration of Cu2-xSe NPs. The investigation suggested that when the concentration of Cu2-xSe NPs was less than 5.0 × 10–8 M, the fluorescence of RhB quenched dramatically, whereas the concentration of Cu2-xSe NPs was greater than 5.0 × 10–8 M, the fluorescence of RhB varied slightly (Fig.2A). The main reason was that when the concentration of Cu2-xSe NPs was relatively low, more RhB molecules were free in the solution, and thereby the fluorescence of RhB could not be quenched completely; however, the increasing concentration of Cu2-xSe NPs would result in the dramatic fluorescence quenching. In addition, if the Cu2-xSe NPs concentration was relatively high, most RhB molecules were adsorbed onto the Cu2-xSe NPs surface, thus the fluorescence of RhB could be almost entirely quenched. In this case, the RhB fluorescence intensity change was not such apparent as increasing Cu2-xSe NPs concentration. The RhB fluorescence, quenched by Cu2-xSe NPs, was gradually recovered with the introduction of L-Cys. Moreover, the recovery of RhB fluorescence was proportional to the concentration of L-Cys (Fig.2B). In this way, a turn on-off fluorescent assay was proposed for L-Cys analysis. The relative mechanism was investigated. As shown in Fig.3, the presence of Cu2-xSe NPs shortened the fluorescence
WANG Xue et al. / Chinese Journal of Analytical Chemistry, 2016, 44(10): 1482–1486
Fig.1
Size characterization of Cu2-xSe NPs: (A) SEM; (B) TEM; (C) Particle size distribution
Fig.2 Fluorescence changes of RhB: (A) fluorescence quenching of RhB induced by Cu2-xSe NPs Conditions: RhB, 6.2 × 10–7 M; Cu2-xSe NPs (0‒6), 0, 0.010, 0.025, 0.050, 0.075, 0.100 and 0.150 μM; T, 30 ºC; t, 2 min; pH 4.6; λem, 575 nm; (B) fluorescence recovery of RhB caused by cysteine. Cu2-xSe NPs, 5.0 × 10–8 M; RhB, 6.2 × 10–7 M; T, 30 ºC; t, 2 min; pH 4.6; λem, 575 nm
lifetime of RhB, indicating that the fluorescence quenching of RhB by Cu2-xSe NPs was a dynamic quenching process[11]. When L-Cys was present in the Cu2-xSe NPs-RhB mixture, the fluorescence lifetime of RhB was basically restored to the previous level (Fig.3A). In the process of dynamic fluorescence quenching, the excited state of RhB molecules could return to the ground state by the mechanism of electron transfer or fluorescence resonance energy transfer. As shown in Fig.3B, the maximum absorption of Cu2-xSe NPs was observed at 1045 nm (Fig.3B inset) rather than in the range of 500–700 nm. That is, no overlapping between the absorption of Cu2-xSe NPs and the emission spectrum of RhB occurred, which excluded the possibility of fluorescence resonance energy transfer. Thus, it is speculated that the quenching caused by electron transfer[12]. Under the optimal conditions, the Zeta potential of RhB was
–6.0 mV, and it was changed to –19.8 mV after addition of Cu2-xSe NPs, suggesting that the binding of Cu2-xSeNPs and RhB increased the negative charge of RhB. When L-Cys was added, the Zeta potential was reduced to –10.4 mV, which indicated that L-Cys combined with Cu2-xSe NPs through the thiol group, making the charge on RhB surface expose to enhance the directional polarization of the surrounding molecules, and thus leading to the fluorescence recovery of RhB. 3.3
Optimization of reaction conditions
The optimization of reaction conditions is illustrated in Fig.4. As shown in Fig.4A, the recovery rate of fluorescence increased when pH < 4.6 and decreased afterwards. This was due to that the weak acid condition (pH 4.6) was close to the isoelectric point of L-Cys (pI = 5.07). At this time, L-Cys, as
Fig.3 Quenching mechanism of RhB fluorescence: (A) lifetime change of RhB; (B) absorption spectra of RhB and Cu2-xSe
WANG Xue et al. / Chinese Journal of Analytical Chemistry, 2016, 44(10): 1482–1486
Fig.4 Optimization of conditions: A, pH. RhB, 6.2 × 10–7 M; Cu2-xSe NPs, 5.0 × 10–8 M; L-Cys, 7.0 × 10–7 M; T, 30 oC; t, 2 min. B, reaction time. RhB, 6.2 × 10–7 M; Cu2-xSe NPs, 5.0 × 10–8 M; L-Cys, 7.0 × 10–7 M; T, 30 oC; pH 4.6. C, Temperature. RhB, 6.2 × 10–7 M; Cu2-xSe NPs, 5.0 × 10–8 M; L-Cys, 7.0 × 10–7 M; pH 4.6; t, 2 min
the zwitterions, was easy to combine with Cu2-xSe NPs through electrostatic interaction, and conducive for Cu2-xSe NPs to bind thiol group of L-Cys[12–14], leading to the enhancement of fluorescence. Therefore, pH 4.6 was selected as the optimum acidity condition. Figure 4B shows the effect of reaction time on fluorescence recovery efficiency. The fluorescence of RhB decreased significantly when Cu2-xSe NPs were added, and then recovered in the presence of L-Cys. The fluorescence signals approximately trended to be stable within 2 min, indicating that this work could be developed for the rapid detection of L-Cys. Figure 4C displays the effect of the reaction temperature on fluorescence recovery efficiency. The fluorescence recovery increased when the temperature was less than 30 ºC, and approached to a steady state as the temperature was higher than 30 ºC. Thus, the mixture should be kept at 30 ºC for 2 min before detection. 3.4
Sensitivity and selectivity
Figure 5A illustrates the relation between the fluorescence intensity of RhB and the contration of L-Cys under the optimal conditions. The fluorescence intensity of RhB at 575 nm was proportional to the L-Cys concentrations in the range of 2.5 × 10–7–1.1 × 10–6 M, which could be expressed as F =
378.8 + 783.4c (μM, r = 0.9935). On the basis of this, the L-Cys was determined in this linearity range with a detection limit (3σ/k) of 5.5 × 10–8 M. The inset in Fig.5A demonstrates the fluorescence change of RhB under the irradiation of 365 UV light lamp. With the increasing concentrations of L-Cys, the fluorescence of RhB was gradually enhanced, so that the visual detection of L-Cys could be realized. This proposed turn on-off strategy supplied a high selectivity towards L-Cys detection (Fig.5B) compared with other amino acids and heparin. Herein, non-polar amino acids including proline (Pro) and phenylalanine (Phe), polar-noncharged amino acids including glycine (Gly) and tyrosine (Tyr), polar-positively-charged amino acid histidine (His), polar-negatively-charged amino acids including aspartic acid (Asp) and glutamate (Glu) were investigated. The results showed that the effect of these substances at the same concentration (7 × 10–7 M) on the fluorescence recovery was negligible. However, L-Cys was able to cause the significant recovery fluorescence of RhB, which would be related to the thiol group of L-Cys.
4
Conclusions
In this work, Cu2-xSe NPs could not only quench the fluorescence of RhB, but also bind L-cys to restore the fluorescence of RhB. Based on this characteristic, a turn on-off
Fig.5 A, The sensitivity of the proposed method. Conditions: RhB, 6.2 × 10–7 M; Cu2-xSe NPs, 5.0 × 10–8 M; T, 30 oC; t, 2 min; pH 4.6; λem, 575 nm. B, The selectivity of the proposed method. Conditions: RhB, 6.2 × 10–7 M; Cu2-xSe, 5.0 × 10–8 M; amino acids and heparin, 7.0 × 10–7 M; T, 30 oC; t, 2 min; pH 4.6; λem, 575 nm
WANG Xue et al. / Chinese Journal of Analytical Chemistry, 2016, 44(10): 1482–1486
fluorescence method for measuring L-cys was established. As a novel kind of quencher, Cu2-xSe NPs were much simpler and easier to prepare compared with other methods[15]. In addition, this proposed fluorescence analysis of L-Cys provided a much higher sensitivity in comparison with other nanomaterials[16], and a much faster response in contrast with our previous works[5,9].
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Li Z, Duan X, Bai Y. Chinese J. Anal. Chem., 2006, 34(8): 1149–1152
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Huang J T, Zeng Q L, Yang X X, Wang J. Analyst, 2013, 138(18): 5296–5302
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[10] Lie S Q, Wang D M, Gao M X, Huang C Z. Nanoscale, 2014, 6(17): 10289–10296
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Cite this article as: Chin J Anal Chem, 2016, 44(10), 1482–1486.
RESEARCH PAPER
Determination of L-Cysteine Based on Energy Transfer between Cu2-xSe Nanoparticles and Rhodamine B WANG Xue, YANG Kun-Cheng, MAO Zhi-Yuan, HUANG Cheng-Zhi*, WANG Jian* Key Laboratory on Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
Abstract: The fluorescence of Rhodamine B (RhB) could be quenched by the manner of photo-induced electron transfer with Cu2-xSe nanoparticles as the energy receptor and RhB as the energy donor. However, L-cysteine (L-Cys) was capable of recovering the fluorescence of Rhodamine B, and the recovered fluorescence intensity was proportional to the concentrations of L-Cys. Based on that, a novel method for detecting L-Cys was established. After mixing L-Cys and RhB pretreated by Cu2-xSe nanoparticles at pH 4.6 and 30 oC for 2 min, a linear relationship was obtained between the fluorescence intensity of RhB at 575 nm and the concentrations of L-Cys in the range of 2.5 × 10–7–1.1 × 10–6 M. This method was used for the determination of L-cys with a detection limit (3σ/k) of 5.5 × 10–8 M. The common amino acids presented little interference for the detection of L-Cys. Key Words: Cu2-xSe nanoparticles; L-cysteine; Photo-induced electron transfer
1
Introduction
As one common amino acid in living organisms, L-cysteine (L-Cys) is mainly found in protein and glutathione, which is associated with the physiological functions of nutrition and immunity in animals[1]. Currently, a verity of methods are developed for L-Cys detection, including electrochemistry[2], fluorescence[3,4], light scattering[5,6] and colorimetry[7–9], etc. Our group established the light scattering technique[4] and colorimetric methods[8,9] for L-Cys sensing with gold nanoparticles (AuNPs) and silver nanoparticles as optical probes. Based on the Au-S covalent bond between L-Cys and AuNPs, as well as the electrostatic interaction between COO– and NH4+ among L-Cys molecules, AuNPs aggregated with the enhanced resonance light scattering signals, which was developed for L-Cys sensing. This light scattering technique supplied a sensitive and selective result; however, it would take 25 min to complete the process[5]. Moreover, AuNPs possessed the outstanding catalytic activity, which could be inhibited
when formed aggregates induced by L-Cys, accompanied by a color change from dark blue to light blue. This finding enabled the visual detection of L-Cys with very sensitive result; however, the process took as long as 60 min[9]. Hence, it is highly desirable to develop speedy approaches to detect L-Cys. The non-stoichiometric Cu2-xSe nanoparticles (Cu2-xSe NPs) are a kind of chalcogenide semiconductor copper nanomaterials, which hold remarkable features, such as unique optical properties, adjustable size, and superior thermal properties[10]. The detection principle is presented in Scheme 1. Cu2-xSe NPs act as the energy acceptor to quench the fluorescence of energy donor-Rhodamine B (RhB). However, L-Cys is capable of recovering the quenched fluorescence of RhB. Based on that, a fluorescent method is established for detecting L-Cys. In this work, the energy transfer was mainly attributed to the photo-induced charge transfer. Wherein, RhB was easier to accept electron at excited state, and electron transfer happened between Cu2-xSe and RhB, resulting in the RhB fluorescence quenching.
________________________ Received 27 May 2016; accepted 14 August 2016 *Corresponding author. Email: [email protected]; [email protected] This work was supported by the National Natural Science Foundation of China (No. 21405123), the Fund of Chongqing Fundamental and Advanced Research Project of China (No. cstc2014jcyjA50006), the National Students' Innovative Training Project of Southwest University of China (No. 201610635050) and the College of Pharmaceutical Sciences Innovation and Entrepreneurship Project of Southwest University, China. Copyright © 2016, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(16)60960-X
WANG Xue et al. / Chinese Journal of Analytical Chemistry, 2016, 44(10): 1482–1486
then stored at 4 ºC. 2.2.2
Scheme 1 Schematic diagram of the detection of L-Cysteine with Cu2-xSe NPs
2
Fluorescence detection
Approximately 0.20 mL of RhB (3.1 × 10–6 M), 0.20 mL of Cu2-xSe NPs (2.5 × 10–7 M) and 0.10 mL of buffer (pH 4.6) were added into a 1.5-mL tube. After stirring, L-Cys at different doses was introduced. The resultant mixture was diluted to 1.0 mL with H2O, which was then fully stirred and stored at 30 ºC for 2 min. The fluorescence spectra were scanned on an F-2700 spectrophotometer at excitation wavelength of 540 nm and the slits of 5 nm.
Experimental 3
2.1
3.1 The absorption and fluorescence spectra were scanned with a UV-2600 spectrophotometer (Shimadzu, Japan) and an F-2700 spectrophotometer (Hitachi, Japan), respectively. A FL-TCSPC fluorescence spectrophotometer (Horiba Jobin Yvon Inc., France) provided the fluorescence lifetime of RhB. Both the scanning electron microscopy (SEM) imaging and transmission electron microscopy (TEM) imaging of Cu2-xSe NPs were acquired with an S-4800 scanning electron microscope (Hitachi, Japan). Zeta potential was carried out on a Nanosizer (ZEN3600, Malvern). A HCJ-6D magnetic stirrer (Changzhou, China) and a pHS-3C pH-meter (Chengdu, China) were employed to mix solutions and measure the acidity of solutions, respectively. Cu2-xSe NPs were purified with a H1650W high speed centrifuge (Hunan, China). The solutions of polyvinylpyrrolidone (PVP, Mw 55 kDa), SeO2, Vitamin c (Vc), Cu(CH3COO)2·H2O, RhB and L-Cys were stored at 4 ºC. All reagents were of analytical grade and used as received without further purification. The acidity of solutions was adjusted with citric acid-citrate sodium buffer. Doubly distilled water was used as solvent throughout this work. 2.2 2.2.1
Results and discussion
Instruments and reagents
Experimental methods Preparation of Cu2-xSe NPs
The non-stoichiometric Cu2-xSe NPs were prepared by a simple one-pot method[11]. Firstly, 1.6 mL of PVP (5 g L–1) and 5.5 mL of water were mixed in a round-bottom flask, and then 0.1 mL of SeO2 (0.1 M) and 0.3 mL of Vc (0.2 M) were introduced. After stirring for 10 min with a magnetic stirrer, a mixture of 0.1 mL of Cu(CH3COO)2·H2O (0.2 M) and 0.4 mL of Vc (0.2 M) was dropped into the flask under vigorous stirring. The resultant mixture was allowed to vigorously stir at 30 ºC for 10 h until a green solution was obtained, which was then purified by a 3500-kDa dialysis membrane for 24 h. Finally, Cu2-xSe NPs were further purified with centrifuge and
Characterization of Cu2-xSe NPs
As shown in Fig.1A and Fig.1B, the SEM and TEM images illustrated that the as-prepared Cu2-xSe NPs were well monodispersed and uniform. The size distribution of Cu2-xSe NPs was analyzed with the Nano-Measurer software. The results indicated that the diameters of Cu2-xSe NPs were mainly presented in the range of 62.4–99.2 nm with an average size of 79.2 nm (Fig.1C). 3.2
Detection of cysteine based on the fluorescence change of RhB
As shown in Fig.2A, the fluorescence of RhB at 575 nm was quenched by Cu2-xSe NPs, which was closely related to the concentration of Cu2-xSe NPs. The investigation suggested that when the concentration of Cu2-xSe NPs was less than 5.0 × 10–8 M, the fluorescence of RhB quenched dramatically, whereas the concentration of Cu2-xSe NPs was greater than 5.0 × 10–8 M, the fluorescence of RhB varied slightly (Fig.2A). The main reason was that when the concentration of Cu2-xSe NPs was relatively low, more RhB molecules were free in the solution, and thereby the fluorescence of RhB could not be quenched completely; however, the increasing concentration of Cu2-xSe NPs would result in the dramatic fluorescence quenching. In addition, if the Cu2-xSe NPs concentration was relatively high, most RhB molecules were adsorbed onto the Cu2-xSe NPs surface, thus the fluorescence of RhB could be almost entirely quenched. In this case, the RhB fluorescence intensity change was not such apparent as increasing Cu2-xSe NPs concentration. The RhB fluorescence, quenched by Cu2-xSe NPs, was gradually recovered with the introduction of L-Cys. Moreover, the recovery of RhB fluorescence was proportional to the concentration of L-Cys (Fig.2B). In this way, a turn on-off fluorescent assay was proposed for L-Cys analysis. The relative mechanism was investigated. As shown in Fig.3, the presence of Cu2-xSe NPs shortened the fluorescence
WANG Xue et al. / Chinese Journal of Analytical Chemistry, 2016, 44(10): 1482–1486
Fig.1
Size characterization of Cu2-xSe NPs: (A) SEM; (B) TEM; (C) Particle size distribution
Fig.2 Fluorescence changes of RhB: (A) fluorescence quenching of RhB induced by Cu2-xSe NPs Conditions: RhB, 6.2 × 10–7 M; Cu2-xSe NPs (0‒6), 0, 0.010, 0.025, 0.050, 0.075, 0.100 and 0.150 μM; T, 30 ºC; t, 2 min; pH 4.6; λem, 575 nm; (B) fluorescence recovery of RhB caused by cysteine. Cu2-xSe NPs, 5.0 × 10–8 M; RhB, 6.2 × 10–7 M; T, 30 ºC; t, 2 min; pH 4.6; λem, 575 nm
lifetime of RhB, indicating that the fluorescence quenching of RhB by Cu2-xSe NPs was a dynamic quenching process[11]. When L-Cys was present in the Cu2-xSe NPs-RhB mixture, the fluorescence lifetime of RhB was basically restored to the previous level (Fig.3A). In the process of dynamic fluorescence quenching, the excited state of RhB molecules could return to the ground state by the mechanism of electron transfer or fluorescence resonance energy transfer. As shown in Fig.3B, the maximum absorption of Cu2-xSe NPs was observed at 1045 nm (Fig.3B inset) rather than in the range of 500–700 nm. That is, no overlapping between the absorption of Cu2-xSe NPs and the emission spectrum of RhB occurred, which excluded the possibility of fluorescence resonance energy transfer. Thus, it is speculated that the quenching caused by electron transfer[12]. Under the optimal conditions, the Zeta potential of RhB was
–6.0 mV, and it was changed to –19.8 mV after addition of Cu2-xSe NPs, suggesting that the binding of Cu2-xSeNPs and RhB increased the negative charge of RhB. When L-Cys was added, the Zeta potential was reduced to –10.4 mV, which indicated that L-Cys combined with Cu2-xSe NPs through the thiol group, making the charge on RhB surface expose to enhance the directional polarization of the surrounding molecules, and thus leading to the fluorescence recovery of RhB. 3.3
Optimization of reaction conditions
The optimization of reaction conditions is illustrated in Fig.4. As shown in Fig.4A, the recovery rate of fluorescence increased when pH < 4.6 and decreased afterwards. This was due to that the weak acid condition (pH 4.6) was close to the isoelectric point of L-Cys (pI = 5.07). At this time, L-Cys, as
Fig.3 Quenching mechanism of RhB fluorescence: (A) lifetime change of RhB; (B) absorption spectra of RhB and Cu2-xSe
WANG Xue et al. / Chinese Journal of Analytical Chemistry, 2016, 44(10): 1482–1486
Fig.4 Optimization of conditions: A, pH. RhB, 6.2 × 10–7 M; Cu2-xSe NPs, 5.0 × 10–8 M; L-Cys, 7.0 × 10–7 M; T, 30 oC; t, 2 min. B, reaction time. RhB, 6.2 × 10–7 M; Cu2-xSe NPs, 5.0 × 10–8 M; L-Cys, 7.0 × 10–7 M; T, 30 oC; pH 4.6. C, Temperature. RhB, 6.2 × 10–7 M; Cu2-xSe NPs, 5.0 × 10–8 M; L-Cys, 7.0 × 10–7 M; pH 4.6; t, 2 min
the zwitterions, was easy to combine with Cu2-xSe NPs through electrostatic interaction, and conducive for Cu2-xSe NPs to bind thiol group of L-Cys[12–14], leading to the enhancement of fluorescence. Therefore, pH 4.6 was selected as the optimum acidity condition. Figure 4B shows the effect of reaction time on fluorescence recovery efficiency. The fluorescence of RhB decreased significantly when Cu2-xSe NPs were added, and then recovered in the presence of L-Cys. The fluorescence signals approximately trended to be stable within 2 min, indicating that this work could be developed for the rapid detection of L-Cys. Figure 4C displays the effect of the reaction temperature on fluorescence recovery efficiency. The fluorescence recovery increased when the temperature was less than 30 ºC, and approached to a steady state as the temperature was higher than 30 ºC. Thus, the mixture should be kept at 30 ºC for 2 min before detection. 3.4
Sensitivity and selectivity
Figure 5A illustrates the relation between the fluorescence intensity of RhB and the contration of L-Cys under the optimal conditions. The fluorescence intensity of RhB at 575 nm was proportional to the L-Cys concentrations in the range of 2.5 × 10–7–1.1 × 10–6 M, which could be expressed as F =
378.8 + 783.4c (μM, r = 0.9935). On the basis of this, the L-Cys was determined in this linearity range with a detection limit (3σ/k) of 5.5 × 10–8 M. The inset in Fig.5A demonstrates the fluorescence change of RhB under the irradiation of 365 UV light lamp. With the increasing concentrations of L-Cys, the fluorescence of RhB was gradually enhanced, so that the visual detection of L-Cys could be realized. This proposed turn on-off strategy supplied a high selectivity towards L-Cys detection (Fig.5B) compared with other amino acids and heparin. Herein, non-polar amino acids including proline (Pro) and phenylalanine (Phe), polar-noncharged amino acids including glycine (Gly) and tyrosine (Tyr), polar-positively-charged amino acid histidine (His), polar-negatively-charged amino acids including aspartic acid (Asp) and glutamate (Glu) were investigated. The results showed that the effect of these substances at the same concentration (7 × 10–7 M) on the fluorescence recovery was negligible. However, L-Cys was able to cause the significant recovery fluorescence of RhB, which would be related to the thiol group of L-Cys.
4
Conclusions
In this work, Cu2-xSe NPs could not only quench the fluorescence of RhB, but also bind L-cys to restore the fluorescence of RhB. Based on this characteristic, a turn on-off
Fig.5 A, The sensitivity of the proposed method. Conditions: RhB, 6.2 × 10–7 M; Cu2-xSe NPs, 5.0 × 10–8 M; T, 30 oC; t, 2 min; pH 4.6; λem, 575 nm. B, The selectivity of the proposed method. Conditions: RhB, 6.2 × 10–7 M; Cu2-xSe, 5.0 × 10–8 M; amino acids and heparin, 7.0 × 10–7 M; T, 30 oC; t, 2 min; pH 4.6; λem, 575 nm
WANG Xue et al. / Chinese Journal of Analytical Chemistry, 2016, 44(10): 1482–1486
fluorescence method for measuring L-cys was established. As a novel kind of quencher, Cu2-xSe NPs were much simpler and easier to prepare compared with other methods[15]. In addition, this proposed fluorescence analysis of L-Cys provided a much higher sensitivity in comparison with other nanomaterials[16], and a much faster response in contrast with our previous works[5,9].
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