Analytica Chimica Acta 850 (2014) 71–77
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A sensitive and selective detection method for thiol compounds using novel fluorescence probe Li-Qing Zheng, Ying Li, Xiao-Dong Yu *, Jing-Juan Xu, Hong-Yuan Chen State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, PR China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A sensitive and selective detection method for thiol compounds using novel fluorescent probe was developed. The fluorescence quenching of the synthesized probe was based on the resonance energy fluorescence transfer (FRET) effect. The synthesized probe containing a disulfide bond which can selectively react with thiol compounds by the thiol-disulfide exchange reaction. This method was successfully utilized to analyze Cys in the compound amino acid injection.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 July 2014 Received in revised form 29 August 2014 Accepted 4 September 2014 Available online 18 September 2014
In this work, a sensitive and selective detection method based on fluorescence resonance energy transfer (FRET) was developed for analyzing thiol compounds by using a novel fluorescent probe. The new fluorescent probe contains a disulfide bond which selectively reacts with nucleophilic thiolate through the thiol-disulfide exchange reaction. An obvious fluorescence recovery can be observed upon addition of the thiol compound in the fluorescent probe solution due to the thiol-disulfide exchange reaction and the destruction of FRET. This novel probe was successfully used to determine dithiothreitol (DTT), glutathione (GSH) and cysteine (Cys). The limits of detection (LOD) were 2.0 mM for DTT, 0.6 mM for GSH, and 0.8 mM for Cys. This new detection method was further investigated in the analysis of compound amino acid injection. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Fluorescence resonance energy transfer Thiol Dithiothreitol Glutathione Cysteine
1. Introduction Thiol compounds, such as dithiothreitol (DTT), glutathione (GSH) and cysteine (Cys), are commonly used in the reduction of the disulfide bonds of proteins. In biological systems, thiol
* Corresponding author. Tel.: +86 2583592774; fax: +86 2583 592774. E-mail address: [email protected] (J.-J. Xu). http://dx.doi.org/10.1016/j.aca.2014.09.004 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
compounds play important roles in maintaining the biological redox homeostasis through the exchange reaction between thiols and disulfide bonds [1–4]. DTT is the most widely used dithiol reductant for protein disulfides [5]. However, DTT is a highly toxic substance [6]. GSH contributes to maintaining the normal functions of the immune system. It is an essential endogenous antioxidant which has often been used for detoxification and protecting the cell membrane from being oxidized [7–9]. Abnormal GSH levels often relate to some serious disease [10,11]. It has been
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demonstrated that a low GSH level leads to the development of autism in children [12]. Cys is one of the twenty amino acids used for protein biosynthesis. Cys deficiency is involved in hematopoiesis decrease, leukocyte loss, skin lesions, and weakness [13–15]. Elevated levels of Cys have been associated with neurotoxicity [16,17]. Therefore, it is very important to precisely measure these thiol compounds. The reported thiol detection methods include high-performance liquid chromatographic (HPLC) [18,19], liquid chromatography–mass spectrometry (LC–MS) [20], electrochemistry [21,22], colorimetric assays [23,24] and chemiluminescence [25] etc. Some of these methods require expensive equipment, complicated operating steps and are usually time-consuming. However, there are lots of concerns on fluorometric methods for thiols determination owing to its simplicity and sensitivity [26–30]. In recent years, a number of novel thiol sensing strategies based on the fluorescence resonance energy transfer (FRET) have been developed [31–35], which have largely improved the sensitivity of thiol detection. FRET will cause the quenching of donor fluorescence whereby an excited state donor D (usually a fluorophore) transfers energy to a proximal ground state acceptor A through long-range dipole–dipole interactions [36]. FRET usually occurs over distances comparable to the dimensions of most biological macromolecules, that is, about 10–100 Å [37]. Among the reported thiol sensing methods based on FRET, many fluorescence turn-off detection modes could considerably increase the likelihood of false positive signals [38]. Therefore, a sensitive and selective fluorescence turn-on thiol sensing technique based on FRET is of great interest. A variety of sensing methods based on the thiol-disulfide exchange reactions for determination of thiol and disulfide redox state of proteins have been developed [39–43]. In the thioldisulfide exchange reaction, a nucleophilic thiolate attacks one of the two sulfur atoms of the target disulfide bond and then generate a new disulfide bond and another thiolate leaving group [5]. The exchange reaction between thiol and disulfide bond possesses high specificity in sensing of thiol compound [28]. In this work, we designed a novel fluorescent probe for the detection of thiols based on FRET. The synthesis of probe was simple and the distance between two FITC groups in the probe was short which is in favor of high FRET quenching efficiency. The strategy is illustrated in Scheme 1. The probe was synthesized by the reaction of the NQCQS group of fluorescein isothiocyanate (FITC) with the amino groups of cystamine dihydrochloride. The two FITC groups of probe molecule are close to each other, causing a rapid quench of the fluorescence intensity at 517 nm of probe due to the FRET. When a thiol compound is added into the probe solution, the nucleophilic thiolate reacts with the disulfide bond in the probe molecule and generates a new disulfide which separates the two FITC groups. As a result, the fluorescence intensity at 517 nm of probe solution dramatically recovers due to the destruction of FRET. The new probe is readily soluble in water
allowing the sensing to be performed in buffer solutions. Successful application of this new probe in determining Cys concentration in a compound amino acid injection has been demonstrated. 2. Experimental 2.1. Chemicals Fluorescein isothiocyanate (FITC), cystamine dihydrochloride, dithiothreitol (DTT), cysteine (Cys), glutathione (GSH), alanine, glycine, glutamine, cystine, serine and other amino acid were purchased from Sigma–Aldrich (Milwaukee, WI). Sodium borohydride (NaBH4), ascorbic acid, methanol, triethylamine, sodium carbonate, sodium bicarbonate, deuterium oxide (D2O), sodium hydrogen sulfite (NaHSO3) and acetic acid were purchased from Nanjing Chemical Reagent Co. Ltd. (Nanjing, China). Acetonitrile was purchased from Merck (New Jersey State, USA). Compound amino acid injection was purchased from Mitsubishi pharmaceuticals company (Guangzhou, China). All the chemicals were analytical-grade reagents except that acetonitrile was chromatographically pure. These chemicals were used without further purification. The water used throughout all experiments was purified by an Elix 5 Pure Water System of Millipore (Billerica, USA). 2.2. Apparatus The fluorescence spectra were performed on a Shimadzu RF5301PC fluorescence spectrophotometer. LC–MS analyses were carried out with an Agilent 1290 Infinity LC/6460 QQQ MS system. 1 H NMR spectrum was measured on a Bruker-600 NMR spectrometer with tetramethylsilane (TMS) as internal standard. Purification of the synthesized probe was performed with Waters 2998 preparative chromatograph system containing a Bridge preparative column (10 250 mm, 5 mm ODS). 2.3. Synthesis of probe FITC (250.0 mg, 0.64 mmol) and cystamine dihydrochloride (67.2 mg, 0.30 mmol) were dissolved in 5 mL methanol/triethylamine (v:v = 100:1), respectively. The two solutions were mixed in the flask. The mixture was heated to 35 C for 5 h under stirring and dark condition, and then the solvent was removed under reduced pressure. The residue was separated by preparative chromatograph with a Bridge preparative column. The mobile phase used in the preparative chromatograph consist of acetonitrile and ultrapure water (v:v = 6:4, 1% acetic acid). The flow rate was 5 mL min 1 and UV detection was performed at 254 nm. 1H NMR (600 MHz, D2O/H2O = 1:9): 1.85 (s, 1H), 2.98 (t, 2H), 3.89 (s, 2H), 6.33 (s, 3H), 6.53 (d, 2H), 6.92 (d, 2H), 7.32 (d, 1H), 7.50 (m, 1H), 3.29 (s, 1H) (Fig. S2).
Scheme 1. Schematic illustration of the mechanism of thiol sensing based on FRET.
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2.4. Fluorometric assay
3.2. Response of probe to thiol compounds
The effects of solution pH, the concentration of probe, the reaction time between thiol compound and probe on fluorescence intensity of probe solution were discussed. Under the optimized conditions, a given concentration of DTT, GSH or Cys was added into 300 mL of 10 5 M probe solution at room temperature. The fluorescence spectra were recorded 16 min after the addition of thiols. For all measurements, the excitation wavelength was set at 493 nm and the excitation and emission slit width were set at 1.5 nm.
The synthesized probe turned out to be highly sensitive to thiol compounds. As shown in Fig. 2, the fluorescence intensity at 517 nm of probe solution was much smaller than that of FITC solution, which indicated that the fluorescence of probe was effectively quenched. The quenching efficiency [(I0 I)/I0] is about 89%, where I and I0 represent the fluorescence intensity at 517 nm of probe solution and FITC solution, respectively. The fluorescence quenching of probe was ascribed to the intramolecular fluorescence resonance energy transfer (FRET) effect. The probe consists of two FITC molecules joined by a disulfide-containing linker. The linker tethers two fluorescein molecules within their Förster radius and then one group can act as a fluorescence quencher for the other [46]. The distance of two fluorophores in the probe was 20.533 Å when the disulfide-containing linker was fully extended (Fig. S4). The separation of two fluorophores is considerably less than the 44 Å Förster radius for fluorescein [47], so the probe showed strong FRET-mediated quenching. When DTT was added into the probe solution, the fluorescence intensity of the solution dramatically recovered and the color change of the solution could be seen by naked eyes. As shown in Scheme 1, the thiol-disulfide bond exchange reaction destroyed the probe molecule and leaded to the separation of two FITC groups. Thus, the FRET was destructed and the fluorescence intensity of solution increased.
3. Results and discussion 3.1. Characterization of probe Successful synthesis of probe was confirmed by LC–MS. Fig. 1 shows the HPLC chromatograms of FITC (a) and the synthesized probe (b). In curve a, the peaks at tR = 5.16 min and tR = 8.96 min corresponded to fluorescein 5 isothiocyanate and fluorescein 6 isothiocyanate, respectively. In curve b, the peak at tR = 5.68 min corresponded to the synthesized probe and there are almost no absorption was recorded at tR = 5.16 min and tR = 8.96 min. This indicated that FITC had reacted with cystamine dihydrochloride completely. As shown in the LC–MS spectrum of probe, the peak at tR = 5.68 min corresponded to two MS peaks at m/z 931.1 [M + H] and 466.1 [M+ + 2H] (Fig. S1). That means the molecular weight of the synthesized probe is 930.1, which is identical to the calculated molecular weight of the probe (C46H34N4O10S4, 930.1). The FT-IR spectra of FITC and probe provided more evidence of the successful synthesis of probe. As shown in Fig. S2, the characteristic absorption band around 2040 cm 1 of NQCQS groups disappeared in curve b, which indicated that the isothiocyanate group of FITC had reacted with the amine groups of cystamine dihydrochloride completely [44]. What is more, the characteristic peaks of FITC in the region of 1110–1590 cm 1 still exist in curve b. 1 H NMR study also demonstrated the successful synthesis of probe (Fig. S3). The chemical shifts of amido bond analog group [ CQS(NH) ] in the probe molecule was observed at 3.29 ppm [45]. The small NMR response signal of this group was ascribed to the replacement of hydrogen by deuterium.
Fig. 1. The HPLC chromatograms of 10 4 M FITC and probe. Chromatographic conditions: the mobile phase consists of acetonitrile and ultrapure water (v:v = 6:4, 1% acetic acid). The flow rate was 1 mL min 1 and UV detection was performed at 254 nm.
3.3. Optimization of the detection conditions for thiol compounds In order to acquire the best sensing response to thiol compounds, the conditions including the solution pH, probe concentration and the reaction time between thiol compound and probe were optimized. During the optimization process, DTT was used as the model thiol compound. 3.3.1. Solution pH Since the fluorescence of FITC and probe are pH-dependent [48], the optimization of solution pH for fluorescence sensing was carried out. Na2CO3/NaHCO3 buffer solution (10 mM, pH 7.4, 8.0, 9.0, 9.68, 10.0) were used to select the best assay solution. As shown in Fig. 3, the probe solution exhibited stable fluorescence intensity under different pH values. After the addition of 20 mM DTT into the probe solution, the fluorescence intensity of probe solution increased due to the destruction of FRET. The maximum fluorescence intensity was found at pH 9.0. To quantify the effect of
Fig. 2. Fluorescence spectra of 10 5 M FITC (a), 10 5 M probe (b) and 10 5 M probe solution with the addition of 50 mM DTT (c). The excitation wavelength was 493 nm. Inset: corresponding photographs of solutions taken under visible light.
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Fig. 3. Fluorescence intensity of the probe solution before (a) and after (b) the addition of 20 mM DTT against pH. Conditions: Na2CO3/NaHCO3 buffer; the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; 10 mM probe solution.
the destruction of FRET, the emission recovery efficiency was measured. The emission recovery efficiency was calculated by (I I0)/I0, where I and I0 represented the fluorescence intensity at 517 nm of the probe solution in the presence and absence of DTT. The higher the emission recovery efficiency, the higher the sensitivity of fluorescence determination. The maximum emission recovery efficiency of the probe solution was obtained at pH 9.0 (Fig. S5). Thus, pH 9.0 was chosen as the optimal pH for further experiments. 3.3.2. The concentration of probe solution The optimal concentration of probe solution was investigated by measuring the emission recovery efficiency of probe solution in the presence of 20 mM DTT. The result was shown in Fig. 4. When the concentration of probe increased continuously, the emission recovery efficiency tremendously increased and then began to decrease. This indicated that the sensitivity of probe to thiol compounds is related to the concentration of probe. The maximum emission recovery efficiency was observed in 10 mM probe solution. This suggested that the FRET of the probe is concentrationdependent. To verify this suggestion, the relationship between fluorescence quenching efficiency and the concentration of probe was studied. As shown in Fig. 5, the fluorescence quenching efficiency of probe increased with increasing the concentration of
Fig. 4. Emission recovery efficiency [(I I0)/I0] of the probe solution with the addition of 20 mM DTT against the concentration of probe. I and I0 represent the fluorescence intensity at 517 nm of the probe solution in the presence and absence of DTT. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; pH 9.0.
Fig. 5. Fluorescence quenching efficiency [(I0 I)/I0] of the probe solution against concentration. I and I0 represent the fluorescence intensity at 517 nm of probe solution and FITC, respectively. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; pH 9.0.
probe up to 10 mM, and then reached to a platform; finally, it reduced with further increasing the concentration of probe. When the concentration of probe was low, the distance between two FITC groups in the probe molecule was long due to the solvent effect. So the FRET effect was weak and the fluorescence quenching efficiency of probe was low. When the concentration of probe increased, the two FITC groups in the probe molecule got closer. The FRET effect became stronger and the fluorescence quenching efficiency of probe increased. When the concentration of probe was in the range of 10–50 mM, the fluorescence quenching efficiencies were almost the same value. Further increasing the concentration of probe, the fluorescence quenching efficiency decreased because the selfquenching effect of FITC became dominated (Fig. S6). Therefore, 10 mM probe solution was chosen as the optimal experiment condition. 3.3.3. Reaction time between thiol compound and probe To investigate when the thiol-disulfide bond exchange reaction will reach equilibrium, the time-dependent fluorescence spectra of probe solution upon the addition of DTT, GSH or Cys were recorded. The results were displayed in Fig. 6. When the reaction time between thiol compounds and probe was prolonged, the fluorescence intensity increased and leveled off to a saturation value after ca. 16 min. Therefore, all the fluorescence measurements were carried out after the addition of the analyte for 16 min.
Fig. 6. Time-dependent fluorescence intensity changes of probe solution with addition of 10 mM DTT, GSH and Cys respectively. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; 10 mM probe solution; pH 9.0.
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3.4. Sensitivity of thiols detection
Fig. 7. Fluorescence spectra of probe solution in the presence of different concentrations of DTT. Inset: plot of I/I0 against concentration of DTT. In the picture, a–i corresponded to the addition of 0, 2 10 6 M, 4 10 6 M, 6 10 6 M, 8 10 6 M, 1 10 5 M, 2 10 5 M, 3 10 5 M, 4 10 5 M. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; 10 mM probe solution; pH 9.0.
The sensitivity of thiols detection was evaluated under the optimized conditions. The results were displayed in Figs. 7–9 When the concentration of DTT, GSH and Cys increased in the probe solution, the fluorescence intensity of probe solution increased accordingly. The insets in the Figs. 7–9 show good linear correlation between I/I0 and the concentration of thiol compounds. I and I0 represent the fluorescence intensity of probe solution at 517 nm in the presence and absence of thiol compound, respectively. The linear range of detection were 4–40 mM for DTT, 0.6–3 mM for GSH and 1–10 mM for Cys. The linear regression equation for DTT, GSH and Cys were Y = 1.2387 + 0.0685C (R = 0.997), Y = 1.1612 + 0.2373C (R = 0.995) and Y = 1.1214 + 0.1021C (R = 0.992), respectively. Y is I/I0 and C is the concentration of thiols in mM. The limits of detection (LOD) were 2.0 mM for DTT, 0.6 mM for GSH, and 0.8 mM for Cys. 3.5. Selectivity of thiols detection The selectivity of thiols detection was studied. The responses to the probe of some frequently-used reducing reagents and amino acids (alanine, glycine, glutamine, serine and cystine) were recorded. As shown in Fig. 10, none of these reducing reagents and amino acids could induce noticeable fluorescence recovery of probe solution except for the compounds containing free thiol group. The results indicated that the probe could detect the thiol compounds specifically. 3.6. Determination of thiols in real sample
Fig. 8. Fluorescence spectra of probe solution in the presence of different concentrations of GSH. Inset: plot of I/I0 against concentration of GSH. In the picture, a–f corresponded to the addition of 0, 6 10 7 M, 8 10 7 M,1 10 6 M, 2 10 6 M, 3 10 6 M. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; 10 mM probe solution; pH 9.0.
Fig. 9. Fluorescence spectra of probe solution in the presence of different concentrations of Cys. Inset: plot of I/I0 against concentration of Cys. In the picture, a–h corresponded to the addition of 0, 8 10 7 M, 1 10 6 M, 2 10 6 M, 4 10 6 M, 6 10 6 M, 8 10 6 M, 1 10 5 M. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; 10 mM probe solution; pH 9.0.
In the real sample assays, Cys in the compound amino acid injection was determined by the synthesized probe. The effects of different amino acids and NaHSO3 in the injection on the detection of Cys had been investigated first. The fluorescence intensity of probe solutions containing 3 mM Cys and the same solution with addition of 100 mM different amino acids or NaHSO3 were recorded. As shown in Fig. 11, the existence of different amino acids and NaHSO3 had little interference in the fluorescence response of probe to Cys. Therefore, the probe was able to detect Cys of the injection selectively with the complicated background. The fluorescence spectra of the probe solution with the addition of real sample and different Cys standard solution were displayed in Fig. 12. When the 1000-fold diluted sample was added into the
Fig. 10. Emission recovery efficiency [(I I0)/I0] of the probe solution with the addition of 50 mM various reducing reagents and amino acids. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; 10 mM probe solution; pH 9.0.
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destruction of FRET. This novel probe was used to detect DTT, GSH and Cys. Good linear detection range and low detection limits for DTT, GSH and Cys were obtained. This sensing method was successfully utilized to determine the concentration of Cys in compound amino acid injection. Acknowledgments This work was supported by the National Instrumentation Program of China (2011YQ17006711), the Major Program of NSFC (21190044), the NSFC Scientific Equipment Joint Fund Project (11179004) and NSFC Innovative Research Group Project (21121091). Appendix A. Supplementary data Fig. 11. Fluorescence intensity of probe solutions containing 3 mM Cys and the same solution with addition of 100 mM different amino acids and NaHSO3. Conditions:
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.09.004. Reference
Fig. 12. Fluorescence spectra of probe solution with addition of real sample. Inset: plot of I/I0 against concentration of Cys standard solution. In the picture, a–e corresponded to the addition of 0, 1 10 6 M, 2 10 6 M, 4 10 6 M of Cys in probe solution containing 1000-fold diluted real sample. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; 10 mM probe solution; pH 9.0.
probe solution, the fluorescence intensity of the probe solution immediately increased. By adding different concentration of Cys into the above solution, a linear enhancement of the fluorescence intensity versus the concentration of Cys was obtained. The linear regression equation was Y = 1.356 + 0.0900C (R = 0.995), where Y is I/I0 and C is the concentration of Cys in mM. The recoveries of Cys were measured to be in the range of 96.7–123%. According to the fluorescence recovery of probe solution, the calculated concentration of Cys was about 3.8 10 3 M in the compound amino acid injection sample, which was consistent with the specification of the injection. 4. Conclusions In the paper, a novel fluorescent probe which can sensitively and selectively respond to thiol compounds was synthesized. The fluorescence intensity of the probe solution was considerably weak via FRET effect, and it increased upon the addition of thiol compound by the thiol-disulfide bond exchange reaction and the
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A sensitive and selective detection method for thiol compounds using novel fluorescence probe Li-Qing Zheng, Ying Li, Xiao-Dong Yu *, Jing-Juan Xu, Hong-Yuan Chen State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, PR China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A sensitive and selective detection method for thiol compounds using novel fluorescent probe was developed. The fluorescence quenching of the synthesized probe was based on the resonance energy fluorescence transfer (FRET) effect. The synthesized probe containing a disulfide bond which can selectively react with thiol compounds by the thiol-disulfide exchange reaction. This method was successfully utilized to analyze Cys in the compound amino acid injection.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 July 2014 Received in revised form 29 August 2014 Accepted 4 September 2014 Available online 18 September 2014
In this work, a sensitive and selective detection method based on fluorescence resonance energy transfer (FRET) was developed for analyzing thiol compounds by using a novel fluorescent probe. The new fluorescent probe contains a disulfide bond which selectively reacts with nucleophilic thiolate through the thiol-disulfide exchange reaction. An obvious fluorescence recovery can be observed upon addition of the thiol compound in the fluorescent probe solution due to the thiol-disulfide exchange reaction and the destruction of FRET. This novel probe was successfully used to determine dithiothreitol (DTT), glutathione (GSH) and cysteine (Cys). The limits of detection (LOD) were 2.0 mM for DTT, 0.6 mM for GSH, and 0.8 mM for Cys. This new detection method was further investigated in the analysis of compound amino acid injection. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Fluorescence resonance energy transfer Thiol Dithiothreitol Glutathione Cysteine
1. Introduction Thiol compounds, such as dithiothreitol (DTT), glutathione (GSH) and cysteine (Cys), are commonly used in the reduction of the disulfide bonds of proteins. In biological systems, thiol
* Corresponding author. Tel.: +86 2583592774; fax: +86 2583 592774. E-mail address: [email protected] (J.-J. Xu). http://dx.doi.org/10.1016/j.aca.2014.09.004 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
compounds play important roles in maintaining the biological redox homeostasis through the exchange reaction between thiols and disulfide bonds [1–4]. DTT is the most widely used dithiol reductant for protein disulfides [5]. However, DTT is a highly toxic substance [6]. GSH contributes to maintaining the normal functions of the immune system. It is an essential endogenous antioxidant which has often been used for detoxification and protecting the cell membrane from being oxidized [7–9]. Abnormal GSH levels often relate to some serious disease [10,11]. It has been
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demonstrated that a low GSH level leads to the development of autism in children [12]. Cys is one of the twenty amino acids used for protein biosynthesis. Cys deficiency is involved in hematopoiesis decrease, leukocyte loss, skin lesions, and weakness [13–15]. Elevated levels of Cys have been associated with neurotoxicity [16,17]. Therefore, it is very important to precisely measure these thiol compounds. The reported thiol detection methods include high-performance liquid chromatographic (HPLC) [18,19], liquid chromatography–mass spectrometry (LC–MS) [20], electrochemistry [21,22], colorimetric assays [23,24] and chemiluminescence [25] etc. Some of these methods require expensive equipment, complicated operating steps and are usually time-consuming. However, there are lots of concerns on fluorometric methods for thiols determination owing to its simplicity and sensitivity [26–30]. In recent years, a number of novel thiol sensing strategies based on the fluorescence resonance energy transfer (FRET) have been developed [31–35], which have largely improved the sensitivity of thiol detection. FRET will cause the quenching of donor fluorescence whereby an excited state donor D (usually a fluorophore) transfers energy to a proximal ground state acceptor A through long-range dipole–dipole interactions [36]. FRET usually occurs over distances comparable to the dimensions of most biological macromolecules, that is, about 10–100 Å [37]. Among the reported thiol sensing methods based on FRET, many fluorescence turn-off detection modes could considerably increase the likelihood of false positive signals [38]. Therefore, a sensitive and selective fluorescence turn-on thiol sensing technique based on FRET is of great interest. A variety of sensing methods based on the thiol-disulfide exchange reactions for determination of thiol and disulfide redox state of proteins have been developed [39–43]. In the thioldisulfide exchange reaction, a nucleophilic thiolate attacks one of the two sulfur atoms of the target disulfide bond and then generate a new disulfide bond and another thiolate leaving group [5]. The exchange reaction between thiol and disulfide bond possesses high specificity in sensing of thiol compound [28]. In this work, we designed a novel fluorescent probe for the detection of thiols based on FRET. The synthesis of probe was simple and the distance between two FITC groups in the probe was short which is in favor of high FRET quenching efficiency. The strategy is illustrated in Scheme 1. The probe was synthesized by the reaction of the NQCQS group of fluorescein isothiocyanate (FITC) with the amino groups of cystamine dihydrochloride. The two FITC groups of probe molecule are close to each other, causing a rapid quench of the fluorescence intensity at 517 nm of probe due to the FRET. When a thiol compound is added into the probe solution, the nucleophilic thiolate reacts with the disulfide bond in the probe molecule and generates a new disulfide which separates the two FITC groups. As a result, the fluorescence intensity at 517 nm of probe solution dramatically recovers due to the destruction of FRET. The new probe is readily soluble in water
allowing the sensing to be performed in buffer solutions. Successful application of this new probe in determining Cys concentration in a compound amino acid injection has been demonstrated. 2. Experimental 2.1. Chemicals Fluorescein isothiocyanate (FITC), cystamine dihydrochloride, dithiothreitol (DTT), cysteine (Cys), glutathione (GSH), alanine, glycine, glutamine, cystine, serine and other amino acid were purchased from Sigma–Aldrich (Milwaukee, WI). Sodium borohydride (NaBH4), ascorbic acid, methanol, triethylamine, sodium carbonate, sodium bicarbonate, deuterium oxide (D2O), sodium hydrogen sulfite (NaHSO3) and acetic acid were purchased from Nanjing Chemical Reagent Co. Ltd. (Nanjing, China). Acetonitrile was purchased from Merck (New Jersey State, USA). Compound amino acid injection was purchased from Mitsubishi pharmaceuticals company (Guangzhou, China). All the chemicals were analytical-grade reagents except that acetonitrile was chromatographically pure. These chemicals were used without further purification. The water used throughout all experiments was purified by an Elix 5 Pure Water System of Millipore (Billerica, USA). 2.2. Apparatus The fluorescence spectra were performed on a Shimadzu RF5301PC fluorescence spectrophotometer. LC–MS analyses were carried out with an Agilent 1290 Infinity LC/6460 QQQ MS system. 1 H NMR spectrum was measured on a Bruker-600 NMR spectrometer with tetramethylsilane (TMS) as internal standard. Purification of the synthesized probe was performed with Waters 2998 preparative chromatograph system containing a Bridge preparative column (10 250 mm, 5 mm ODS). 2.3. Synthesis of probe FITC (250.0 mg, 0.64 mmol) and cystamine dihydrochloride (67.2 mg, 0.30 mmol) were dissolved in 5 mL methanol/triethylamine (v:v = 100:1), respectively. The two solutions were mixed in the flask. The mixture was heated to 35 C for 5 h under stirring and dark condition, and then the solvent was removed under reduced pressure. The residue was separated by preparative chromatograph with a Bridge preparative column. The mobile phase used in the preparative chromatograph consist of acetonitrile and ultrapure water (v:v = 6:4, 1% acetic acid). The flow rate was 5 mL min 1 and UV detection was performed at 254 nm. 1H NMR (600 MHz, D2O/H2O = 1:9): 1.85 (s, 1H), 2.98 (t, 2H), 3.89 (s, 2H), 6.33 (s, 3H), 6.53 (d, 2H), 6.92 (d, 2H), 7.32 (d, 1H), 7.50 (m, 1H), 3.29 (s, 1H) (Fig. S2).
Scheme 1. Schematic illustration of the mechanism of thiol sensing based on FRET.
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2.4. Fluorometric assay
3.2. Response of probe to thiol compounds
The effects of solution pH, the concentration of probe, the reaction time between thiol compound and probe on fluorescence intensity of probe solution were discussed. Under the optimized conditions, a given concentration of DTT, GSH or Cys was added into 300 mL of 10 5 M probe solution at room temperature. The fluorescence spectra were recorded 16 min after the addition of thiols. For all measurements, the excitation wavelength was set at 493 nm and the excitation and emission slit width were set at 1.5 nm.
The synthesized probe turned out to be highly sensitive to thiol compounds. As shown in Fig. 2, the fluorescence intensity at 517 nm of probe solution was much smaller than that of FITC solution, which indicated that the fluorescence of probe was effectively quenched. The quenching efficiency [(I0 I)/I0] is about 89%, where I and I0 represent the fluorescence intensity at 517 nm of probe solution and FITC solution, respectively. The fluorescence quenching of probe was ascribed to the intramolecular fluorescence resonance energy transfer (FRET) effect. The probe consists of two FITC molecules joined by a disulfide-containing linker. The linker tethers two fluorescein molecules within their Förster radius and then one group can act as a fluorescence quencher for the other [46]. The distance of two fluorophores in the probe was 20.533 Å when the disulfide-containing linker was fully extended (Fig. S4). The separation of two fluorophores is considerably less than the 44 Å Förster radius for fluorescein [47], so the probe showed strong FRET-mediated quenching. When DTT was added into the probe solution, the fluorescence intensity of the solution dramatically recovered and the color change of the solution could be seen by naked eyes. As shown in Scheme 1, the thiol-disulfide bond exchange reaction destroyed the probe molecule and leaded to the separation of two FITC groups. Thus, the FRET was destructed and the fluorescence intensity of solution increased.
3. Results and discussion 3.1. Characterization of probe Successful synthesis of probe was confirmed by LC–MS. Fig. 1 shows the HPLC chromatograms of FITC (a) and the synthesized probe (b). In curve a, the peaks at tR = 5.16 min and tR = 8.96 min corresponded to fluorescein 5 isothiocyanate and fluorescein 6 isothiocyanate, respectively. In curve b, the peak at tR = 5.68 min corresponded to the synthesized probe and there are almost no absorption was recorded at tR = 5.16 min and tR = 8.96 min. This indicated that FITC had reacted with cystamine dihydrochloride completely. As shown in the LC–MS spectrum of probe, the peak at tR = 5.68 min corresponded to two MS peaks at m/z 931.1 [M + H] and 466.1 [M+ + 2H] (Fig. S1). That means the molecular weight of the synthesized probe is 930.1, which is identical to the calculated molecular weight of the probe (C46H34N4O10S4, 930.1). The FT-IR spectra of FITC and probe provided more evidence of the successful synthesis of probe. As shown in Fig. S2, the characteristic absorption band around 2040 cm 1 of NQCQS groups disappeared in curve b, which indicated that the isothiocyanate group of FITC had reacted with the amine groups of cystamine dihydrochloride completely [44]. What is more, the characteristic peaks of FITC in the region of 1110–1590 cm 1 still exist in curve b. 1 H NMR study also demonstrated the successful synthesis of probe (Fig. S3). The chemical shifts of amido bond analog group [ CQS(NH) ] in the probe molecule was observed at 3.29 ppm [45]. The small NMR response signal of this group was ascribed to the replacement of hydrogen by deuterium.
Fig. 1. The HPLC chromatograms of 10 4 M FITC and probe. Chromatographic conditions: the mobile phase consists of acetonitrile and ultrapure water (v:v = 6:4, 1% acetic acid). The flow rate was 1 mL min 1 and UV detection was performed at 254 nm.
3.3. Optimization of the detection conditions for thiol compounds In order to acquire the best sensing response to thiol compounds, the conditions including the solution pH, probe concentration and the reaction time between thiol compound and probe were optimized. During the optimization process, DTT was used as the model thiol compound. 3.3.1. Solution pH Since the fluorescence of FITC and probe are pH-dependent [48], the optimization of solution pH for fluorescence sensing was carried out. Na2CO3/NaHCO3 buffer solution (10 mM, pH 7.4, 8.0, 9.0, 9.68, 10.0) were used to select the best assay solution. As shown in Fig. 3, the probe solution exhibited stable fluorescence intensity under different pH values. After the addition of 20 mM DTT into the probe solution, the fluorescence intensity of probe solution increased due to the destruction of FRET. The maximum fluorescence intensity was found at pH 9.0. To quantify the effect of
Fig. 2. Fluorescence spectra of 10 5 M FITC (a), 10 5 M probe (b) and 10 5 M probe solution with the addition of 50 mM DTT (c). The excitation wavelength was 493 nm. Inset: corresponding photographs of solutions taken under visible light.
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Fig. 3. Fluorescence intensity of the probe solution before (a) and after (b) the addition of 20 mM DTT against pH. Conditions: Na2CO3/NaHCO3 buffer; the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; 10 mM probe solution.
the destruction of FRET, the emission recovery efficiency was measured. The emission recovery efficiency was calculated by (I I0)/I0, where I and I0 represented the fluorescence intensity at 517 nm of the probe solution in the presence and absence of DTT. The higher the emission recovery efficiency, the higher the sensitivity of fluorescence determination. The maximum emission recovery efficiency of the probe solution was obtained at pH 9.0 (Fig. S5). Thus, pH 9.0 was chosen as the optimal pH for further experiments. 3.3.2. The concentration of probe solution The optimal concentration of probe solution was investigated by measuring the emission recovery efficiency of probe solution in the presence of 20 mM DTT. The result was shown in Fig. 4. When the concentration of probe increased continuously, the emission recovery efficiency tremendously increased and then began to decrease. This indicated that the sensitivity of probe to thiol compounds is related to the concentration of probe. The maximum emission recovery efficiency was observed in 10 mM probe solution. This suggested that the FRET of the probe is concentrationdependent. To verify this suggestion, the relationship between fluorescence quenching efficiency and the concentration of probe was studied. As shown in Fig. 5, the fluorescence quenching efficiency of probe increased with increasing the concentration of
Fig. 4. Emission recovery efficiency [(I I0)/I0] of the probe solution with the addition of 20 mM DTT against the concentration of probe. I and I0 represent the fluorescence intensity at 517 nm of the probe solution in the presence and absence of DTT. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; pH 9.0.
Fig. 5. Fluorescence quenching efficiency [(I0 I)/I0] of the probe solution against concentration. I and I0 represent the fluorescence intensity at 517 nm of probe solution and FITC, respectively. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; pH 9.0.
probe up to 10 mM, and then reached to a platform; finally, it reduced with further increasing the concentration of probe. When the concentration of probe was low, the distance between two FITC groups in the probe molecule was long due to the solvent effect. So the FRET effect was weak and the fluorescence quenching efficiency of probe was low. When the concentration of probe increased, the two FITC groups in the probe molecule got closer. The FRET effect became stronger and the fluorescence quenching efficiency of probe increased. When the concentration of probe was in the range of 10–50 mM, the fluorescence quenching efficiencies were almost the same value. Further increasing the concentration of probe, the fluorescence quenching efficiency decreased because the selfquenching effect of FITC became dominated (Fig. S6). Therefore, 10 mM probe solution was chosen as the optimal experiment condition. 3.3.3. Reaction time between thiol compound and probe To investigate when the thiol-disulfide bond exchange reaction will reach equilibrium, the time-dependent fluorescence spectra of probe solution upon the addition of DTT, GSH or Cys were recorded. The results were displayed in Fig. 6. When the reaction time between thiol compounds and probe was prolonged, the fluorescence intensity increased and leveled off to a saturation value after ca. 16 min. Therefore, all the fluorescence measurements were carried out after the addition of the analyte for 16 min.
Fig. 6. Time-dependent fluorescence intensity changes of probe solution with addition of 10 mM DTT, GSH and Cys respectively. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; 10 mM probe solution; pH 9.0.
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3.4. Sensitivity of thiols detection
Fig. 7. Fluorescence spectra of probe solution in the presence of different concentrations of DTT. Inset: plot of I/I0 against concentration of DTT. In the picture, a–i corresponded to the addition of 0, 2 10 6 M, 4 10 6 M, 6 10 6 M, 8 10 6 M, 1 10 5 M, 2 10 5 M, 3 10 5 M, 4 10 5 M. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; 10 mM probe solution; pH 9.0.
The sensitivity of thiols detection was evaluated under the optimized conditions. The results were displayed in Figs. 7–9 When the concentration of DTT, GSH and Cys increased in the probe solution, the fluorescence intensity of probe solution increased accordingly. The insets in the Figs. 7–9 show good linear correlation between I/I0 and the concentration of thiol compounds. I and I0 represent the fluorescence intensity of probe solution at 517 nm in the presence and absence of thiol compound, respectively. The linear range of detection were 4–40 mM for DTT, 0.6–3 mM for GSH and 1–10 mM for Cys. The linear regression equation for DTT, GSH and Cys were Y = 1.2387 + 0.0685C (R = 0.997), Y = 1.1612 + 0.2373C (R = 0.995) and Y = 1.1214 + 0.1021C (R = 0.992), respectively. Y is I/I0 and C is the concentration of thiols in mM. The limits of detection (LOD) were 2.0 mM for DTT, 0.6 mM for GSH, and 0.8 mM for Cys. 3.5. Selectivity of thiols detection The selectivity of thiols detection was studied. The responses to the probe of some frequently-used reducing reagents and amino acids (alanine, glycine, glutamine, serine and cystine) were recorded. As shown in Fig. 10, none of these reducing reagents and amino acids could induce noticeable fluorescence recovery of probe solution except for the compounds containing free thiol group. The results indicated that the probe could detect the thiol compounds specifically. 3.6. Determination of thiols in real sample
Fig. 8. Fluorescence spectra of probe solution in the presence of different concentrations of GSH. Inset: plot of I/I0 against concentration of GSH. In the picture, a–f corresponded to the addition of 0, 6 10 7 M, 8 10 7 M,1 10 6 M, 2 10 6 M, 3 10 6 M. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; 10 mM probe solution; pH 9.0.
Fig. 9. Fluorescence spectra of probe solution in the presence of different concentrations of Cys. Inset: plot of I/I0 against concentration of Cys. In the picture, a–h corresponded to the addition of 0, 8 10 7 M, 1 10 6 M, 2 10 6 M, 4 10 6 M, 6 10 6 M, 8 10 6 M, 1 10 5 M. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; 10 mM probe solution; pH 9.0.
In the real sample assays, Cys in the compound amino acid injection was determined by the synthesized probe. The effects of different amino acids and NaHSO3 in the injection on the detection of Cys had been investigated first. The fluorescence intensity of probe solutions containing 3 mM Cys and the same solution with addition of 100 mM different amino acids or NaHSO3 were recorded. As shown in Fig. 11, the existence of different amino acids and NaHSO3 had little interference in the fluorescence response of probe to Cys. Therefore, the probe was able to detect Cys of the injection selectively with the complicated background. The fluorescence spectra of the probe solution with the addition of real sample and different Cys standard solution were displayed in Fig. 12. When the 1000-fold diluted sample was added into the
Fig. 10. Emission recovery efficiency [(I I0)/I0] of the probe solution with the addition of 50 mM various reducing reagents and amino acids. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; 10 mM probe solution; pH 9.0.
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destruction of FRET. This novel probe was used to detect DTT, GSH and Cys. Good linear detection range and low detection limits for DTT, GSH and Cys were obtained. This sensing method was successfully utilized to determine the concentration of Cys in compound amino acid injection. Acknowledgments This work was supported by the National Instrumentation Program of China (2011YQ17006711), the Major Program of NSFC (21190044), the NSFC Scientific Equipment Joint Fund Project (11179004) and NSFC Innovative Research Group Project (21121091). Appendix A. Supplementary data Fig. 11. Fluorescence intensity of probe solutions containing 3 mM Cys and the same solution with addition of 100 mM different amino acids and NaHSO3. Conditions:
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.09.004. Reference
Fig. 12. Fluorescence spectra of probe solution with addition of real sample. Inset: plot of I/I0 against concentration of Cys standard solution. In the picture, a–e corresponded to the addition of 0, 1 10 6 M, 2 10 6 M, 4 10 6 M of Cys in probe solution containing 1000-fold diluted real sample. Conditions: the excitation wavelength was 493 nm; the emission wavelength was 517 nm; reaction time was 16 min; 10 mM probe solution; pH 9.0.
probe solution, the fluorescence intensity of the probe solution immediately increased. By adding different concentration of Cys into the above solution, a linear enhancement of the fluorescence intensity versus the concentration of Cys was obtained. The linear regression equation was Y = 1.356 + 0.0900C (R = 0.995), where Y is I/I0 and C is the concentration of Cys in mM. The recoveries of Cys were measured to be in the range of 96.7–123%. According to the fluorescence recovery of probe solution, the calculated concentration of Cys was about 3.8 10 3 M in the compound amino acid injection sample, which was consistent with the specification of the injection. 4. Conclusions In the paper, a novel fluorescent probe which can sensitively and selectively respond to thiol compounds was synthesized. The fluorescence intensity of the probe solution was considerably weak via FRET effect, and it increased upon the addition of thiol compound by the thiol-disulfide bond exchange reaction and the
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