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Development of a novel rhodamine-type fluorescent probe to determine peroxynitrite
Talanta 57 (2002) 883– 890 www.elsevier.com/locate/talanta
Development of a novel rhodamine-type fluorescent probe to determine peroxynitrite Xiao-Feng Yang, Xiang-Qun Guo *, Yi-Bing Zhao Department of Chemistry and the Key Laboratory of Analytical Sciences of MOE, Xiamen Uni6ersity, Xiamen 361005, Fujian, PR China Received 13 December 2001; received in revised form 20 March 2002; accepted 22 March 2002
Abstract A novel method for the determination of peroxynitrite using rhodamine B hydrazide as a fluorogenic probe is described. The method is based on the oxidation of rhodamine B hydrazide, a colorless, non-fluorescent substance, by peroxynitrite to give rhodamine B-like fluorescence emission. The fluorescence increase is linearly related to the concentration of peroxynitrite in the range of 7.5 × 10 − 8 – 3.0×10 − 6 mol l − 1 with a detection limit of 2.4 × 10 − 8 mol l − 1 (3|). The optimal conditions for the detection of peroxynitrite were evaluated and the possible detection mechanism was also discussed in this paper. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Peroxynitrite; Rhodamine B hydrazide; Fluorimetry; Probe
1. Introduction Peroxynitrite, the product of the combination reaction between nitric oxide (NO) and superox ide radical (O− 2 ) [1], is a potent biological oxidant that promotes oxidative molecular and tissue damage [2,3]. Peroxynitrite formation and reactions are proposed to contribute to the pathogenesis of a series of diseases, including inflammatory processes, ischemia-reperfusion, septic shock and neurodegenerative disorders [4 – 6]. Study of the role of peroxynitrite needs real-time and sensitive probing of peroxynitrite in both pathological and * Corresponding author. Tel.: + 86-592-21-82442; fax: + 86-592-21-88054. E-mail address: [email protected] (X.-Q. Guo).
normal conditions in biological systems, which is indeed extremely difficult because of the low concentration, high activity and elusive nature of peroxynitrite. Peroxynitrite generation is usually measured by UV –visible spectrometry [7], chemiluminescence [8,9], electrochemistry [10], and immunohistochemistry [11]. Two fluorogenic probes, dihydrodichlorofluorescein (DCFH) and dihydrorhodamine-123 (DHR-123), have been widely employed to monitor peroxynitrite production in a variety of systems [12 –15]. The methods are based on the use of chemically reduced, nonfluorescent forms of highly fluorescent dyes such as fluorescein and rhodamine that are oxidized by peroxynitrite to the parent dye molecule, resulting in a dramatic increase in fluorescence intensity.
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Thus, these ‘dihydro’ derivates can serve as fluorogenic probes for monitoring peroxynitrite production in biological systems. Rhodamine derivatives are prized for their great photostability, pH insensitivity over a broad range (low to neutral pH), high quantum yield in aqueous solution and to be excitable at long wavelength, and have been widely used for studying biological systems. In the present study, the characteristic of a non-fluorescent spiro form rhodamine B hydrazide was studied and the results showed that it has a sensitive response to proxynitrite. The reaction of peroxynitrite with rhodamine B hydrazide, a colorless, non-fluorescent compound results in a dramatic increase in fluorescence intensity of the reaction mixture. Based on this phenomenon, a novel fluorescent probe for peroxynitrite was developed. The objectives of the present study are to evaluate the ability of rhodamine B hydrazide as a probe for peroxynitrite and its potential use in biological systems. 2. Experimental
2.1. Chemicals Stock solution of rhodamine B hydrazide (II, 1.0 ×10 − 3 mol l − 1) was prepared by dissolving appropriate amount of II in 30% acetonitrile– water solution. Superoxide dismutase (SOD) (from bovine erythrocytes, 3000 U mg − 1) was obtained from Sigma. A dimethyl sulfoxide (DMSO, 1.0 mol l − 1) solution was prepared. A 1/15 mol l − 1 of KH2PO4 –Na2HPO4 buffer (pH 7.4) was also prepared.
All the reagents were of analytical-reagent grade, and doubly distilled water was used throughout.
2.2. Synthesis of rhodamine B hydrazide (II) A modification of the procedure of Dujols et al. [16] for the synthesis of II was employed. II was synthesized by a one-step reaction of rhodamine B (I) with hydrazine hydrate in methanol (Scheme 1). To a 0.4 g of rhodamine B (I) dissolved in 15 ml of methanol, an excessive hydrazine hydrate (0.5 ml) was added and then the reaction solution was refluxed till the pink color disappeared. After that, the cooled reaction solution was poured into distilled water and extracted with ethyl acetate (6× 25 ml). The combined extracts were dried with sodium sulfate anhydrous, filtered, and then evaporated. The solvent yielded 0.27 g (68%) of II. MS (CI) m/e 457.3, ([M+ H]+); M+, calculated 456.2. 1H NMR (CDCl3): l 7.93 (m, 1H, ArH), 7.45 (m, 2H, ArH), 7.11 (m, 1H, ArH), 6.46 (d, 2H, xanthene-H), 6.42 (d, 2H, xantheneH), 6.29 (dd, 2H, xanthene-H), 3.61 (s, 2H, NH2), 3.34 (q, 8H, NCH2CH3), 1.17 (t, 12H, NCH2CH3).
2.3. Synthesis of peroxynitrite Peroxynitrite was synthesized by the autoxidation of hydroxylamine in alkaline medium. The procedure was as follows: the mixture solution containing 0.01 mol l − 1 of hydroxylamine, 0.5 mol l − 1 of sodium hydroxide and 0.001 mol l − 1 of EDTA, was stirred vigorously in aerobic conditions for about 4–5 h, then some MnO2 powder
Scheme 1.
X.-F. Yang et al. / Talanta 57 (2002) 883–890
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Scheme 2.
was added to the mixture solution to eliminate H2O2 generated in the reaction solution, after that the mixture was filtered and stored at − 18 °C. Peroxynitrite concentration was determined by UV spectrometry at 302 nm (m = 1670 l mol − 1 cm − 1) [17].
2.4. Apparatus The fluorescence spectra and relative fluorescence intensity were measured with a Shimadzu RF-5000 spectrofluorimeter (Kyoto, Japan) with a 10 mm quartz cuvette. The excitation and emission wavelength band-passes were both set at 5 nm. Absorption spectra were obtained on a Beckman DU-7400 Ultraviolet– Visible Spectrophotometer. All pH values were measured with a pH S-301 digital ion meter.
acid (ONOOH), to which NaOH is added immediately to generate the peroxynitrite anion (ONOO−) [18]. However, the elusive nature of ONOOH (t1/2, 1 s) makes the ‘timing’ of both processes (nitrosation and addition of NaOH) the key point for the success of the procedure. Therefore, this method often requires the use of a quenched-flow reactor. Another commonly used method is based on the reaction of H2O2 with alkyl nitrites in alkaline solutions [19]. This method is clean, simple and fast, but alkyl nitrite is not widely available. Hughes et al. had reported that the autoxidation of hydroxylamine in alkaline solutions led to the formation of peroxynitrite as the major product [20]. Hence, we synthesized peroxynitrite based on this reaction (Scheme 2). Fig. 1 shows the typical spectra of the system at
2.5. Procedure In a set of 10 ml-volumetric tubes containing pH 7.4 of phosphate buffer solution, 1.0 ml of II (2.0 ×10 − 5 mol l − 1) and different amount of peroxynitrite were added. The reaction solution was kept at room temperature for 2 min, and then the reaction solution was diluted to the mark with water. The fluorescence intensity of the solution was recorded at 574 nm with the excitation wavelength set at 556 nm.
3. Results and discussion
3.1. Preparation of peroxynitrite There are several methods reported for the synthesis of peroxynitrite. The commonly used method for the synthesis of peroxynitrite is based on the fast nitrosation of H2O2 in acid medium to yield the solution of the unstable peroxynitrous
Fig. 1. Typical reaction spectrum showing the formation of peroxynitrite from the autoxidation of hydroxylamine in alkaline solution. Hydroxylamine, 0.01 mol l − 1; NaOH, 0.5 mol l − 1; EDTA, 0.001 mol l − 1. The reaction was carried out at aerobic condition at room temperature (20 °C), with vigorous stirring. The spectrum was recorded by transferring 2.0 ml of reaction mixture to a 10-ml volumetric tube at different reaction time and diluting to the mark with water. t: (a) 5 min; (b) 20 min; (c) 35 min; (d) 55 min; (e) 75 min; (f) 95 min; (g) 115 min; (h) 135 min; (i) 155 min.
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excitation nor fluorescence emission spectra. However, a mixture of II with peroxynitrite shows apparent spectral characteristics with excitation maximum at 556 nm and fluorescence emission maximum at 574 nm.
3.3. Optimization of the general procedure
Fig. 2. Fluorescence excitation and emission spectra of the system. a, a%, II (2.0 ×10 − 6 mol l − 1); b, b%, II (2.0×10 − 6 mol l − 1)+ peroxynitrite (7.5 × 10 − 7 mol l − 1); c, c%, rhodamine B (2.5 ×10 − 8 mol l − 1). The reaction of peroxynitrite with II was carried out in phosphate buffer (pH 7.4) at room temperature (20 °C) for 2 min. All the spectra were recorded at pH 7.4 of phosphate buffer.
different stages of reaction. As the reaction proceeds, a new band centered on 300– 302 nm, corresponding to ONOO− is formed. EDTA is added as the metal-ion sequestering to the reaction solution to preclude the decomposition of peroxynitrite. The peroxynitrite concentration is estimated from absorbance at 302 nm, nitrate and peroxide scarcely shows absorbance in this region (m= 7 and 40 l mol − 1 cm − 1, respectively) [20]. Moreover, the coexisting species, such as hydrox− ylamine, NO− 3 , NO2 , and H2O2 show no response to the fluorescence increase of the solution of II (see Section 3.6). The present method for the preparation of peroxynitrite is easy to operate, needs no special equipment and only inexpensive and easy available reagents are used.
3.3.1. Effect of reaction time The kinetic characteristics of the proposed detection system were studied. Upon the addition of peroxynitrite to the solution of II in phosphate buffer, the fluorescence intensity of the detection system was recorded as a function of reaction time (as shown in Fig. 3). From Fig. 3, one may see the fluorescence intensity of the detection system increased dramatically in a few seconds and leveled off as the reaction went on, while the fluorescence background of the detection system in the absence of peroxynitrite remained unchanged at the same time. The fluorescence intensity of the detection system reached its maximum value for about 1 min, after that the fluorescence intensity of the detection system remained almost constant. To obtain a high sensitivity and reproducible results, a 2-min reaction time was selected in the following experiment.
3.2. Spectra characteristics II is a colorless, non-fluorescent substance. When peroxynitrite was introduced to the solution of II, a fluorescence emission similar to that of rhodamine B was observed. Fig. 2 shows the fluorescence excitation and emission spectra of II and the mixture of II with peroxynitrite in phosphate solution (pH 7.4). It can be seen that II shows no obvious spectra characteristics, neither
Fig. 3. Kinetic behavior of the present system. (a) II; (b) II+ peroxynitrite (7.5 × 10 − 7 mol l − 1); (c) II + peroxynitrite (1.5 ×10 − 6 mol l − 1). II, 2.0 × 10 − 6 mol l − 1. The fluorescence development and measurement were both carried out in pH 7.4 of phosphate buffer solution.
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concentration of II was up to 1.2× 10 − 6 mol l − 1, therefore, 2.0×10 − 6 mol l − 1 of II was recommended for the subsequent experiment.
Fig. 4. Effect of pH on the fluorescence development reaction of the system. The reaction of II (2.0× 10 − 6 mol l − 1) with peroxynitrite (7.5 ×10 − 7 mol l − 1) was carried out in phosphate buffer with different pH value for 2 min, then pH of the system was adjusted to 7.4 and the fluorescence intensity was recorded.
3.3.2. Effect of pH The effect of pH on the fluorogenic reaction was studied in the range of 5.0– 9.1 in phosphate buffer solution, and the results are shown in Fig. 4. It can be seen that fluorescence increment (DF) of the detection system was increased with pH up to 6.8, remained almost the same in the range of 6.8 – 7.7, and decreased when pH value was above 7.7. Hence, pH 7.4 of phosphate buffer was chosen for fluorogenic reaction in the following study. It is reported that peroxynitrite has a pKa of 6.8 [21], so at pH 9.0, about 99.4% of peroxynitrite is present in the form of ONOO−, while at pH 5.0, about 98.4% of peroxynitrite is present as the conjugate acid, ONOOH. From Fig. 4, it can be seen that the fluorescence increase of the system be observed in a significant degree at pH 5.0 and at 9.0, respectively, indicating that both ONOOH and ONOO− can oxidize II to yield a highly fluorescent product, which is consistent with the oxidizing nature of both species. 3.3.3. Effect of the concentration of II The effect of the concentration of II on DF of the system was studied and the results were shown in Fig. 5. From Fig. 5, it can be seen that DF of the detection system was increased with increasing the concentration of II from 4.0× 10 − 7 to 1.2× 10 − 6 mol l − 1 and remained constant when the
3.3.4. Stability of II The stability of II was studied at different pH values and the experiments showed the fluorescence background of the system varied with pH. In strongly acid media (0.1 mol l − 1 sulfuric acid), the fluorescence background was unstable and increased with time, presumably because II was partly decomposed in strongly acid media and hence a high fluorescence background was recorded, while the fluorescence background of the proposed system was low and stable when the pH of the detection system was ]3. To maintain its stability, II should be kept in neutral or near neutral pH solution, and the water solution of II was able to stand at room temperature for 3 weeks without apparent increase of fluorescence background. (Further experiment on the stability of II with time was not carried out.) 3.4. Possible detection mechanism The oxidation of the various substrates by peroxynitrite (OONO−/ONOOH) can take place via multiple pathways: (i) peroxynitrite may directly
Fig. 5. Effect of the concentration of II on the fluorescence increment (DF) of the system. II (different concentrations) + peroxynitrite (7.5 ×10 − 7 mol l − 1) +phosphate buffer (pH 7.4). The reaction mixture was carried out at room temperature (20 °C) for 2 min, and then the fluorescence intensity was recorded.
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Scheme 3.
oxidize the substrates. (ii) Peroxynitrite may decompose firstly into highly reactive species (OH, NO2), which subsequently oxidizes the substrate or hydroxylates and nitrates aromatic compound. In the present study, the possible mechanism of the oxidation of II by peroxynitrite is studied. Firstly, DMSO (0.1 mol l − 1), a specific scavenger for OH, was introduced to the reaction mixture before the addition of peroxynitrite, and we found the DF of the reaction mixture was almost unchanged, indicating that OH does not contribute to the fluorescence increase of the system. Second, SOD (3 U ml − 1), a specific scavenger for O− 2 , was added to the reaction system, and the fluorescence of the system showed no changes compared with that in the absence of SOD, proving that does not mediate the fluorescence increase of O− 2 the system. The above experiments suggest that the oxidation of II might arise from the peroxynitrite itself, but not its decomposed reactive species. This can be explained that the reaction rate of peroxynitrate with II (k4) was faster than the self-decomposition rate of peroxynitrite via the pathway of 2 and 3 described in Scheme 3 (k2, k3 are 0.017 and 0.8 s − 1, respectively [22]. k4 is still unknown, but according to the literature, which reported the reaction rate of direct oxidation of some compounds by peroxynitrite were 104 l mol − 1 s − 1 [23], we assume that the value of k4 is 104 l mol − 1 s − 1). Third, comparison of the excitation and emission fluorescence spectra of the reaction system with that of authentic rhodamine B shows that they are identical, both having excitation maximum at 556 nm and emission maximum at 574 nm (shown in Fig. 2), indicating that the fluorescent product generated in the reaction
mixture might be rhodamine B. Based on the above experimental results, a possible mechanism of the proposed method is given by Scheme 3.
3.5. Limit of detection, linear concentration range and precision Under the selected conditions given above, the fluorescence increment shows a linear relationship with the concentration of peroxynitrite in the range of 7.5×10 − 8 –3.0×10 − 6 mol l − 1 (r= 0.9991, n= 8). The detection limit is 2.4× 10 − 8 mol l − 1 (3|). The relative standard deviation (n= 7) is 4.1% for 7.5× 10 − 7 mol l − 1 of peroxynitrite.
3.6. Interference study The interferences of the proposed probe for peroxynitrite were studied. A variety of interfering agents were added to the solution of II in pH 7.4 of phosphate buffer, and the response of II to these compounds are listed in Table 1. Interestingly, it was found that as high as 10 − 5 mol l − 1 of Cu2 + did not produce significant fluorescence increment of the detection system described in this paper, which is not contradicted with the fact described by Dujols et al. [16], because the selective response of II to Cu2 + must be carried out in 20% acetonitrile–water solution. If 20% of acetonitrile was added to the solution of II containing Cu2 + , a simultaneous fluorescence increment was observed. Hence, the proposed probe for peroxynitrite without acetonitrile could successfully avoid the interference from Cu2 + .
X.-F. Yang et al. / Talanta 57 (2002) 883–890
4. Conclusion In summary, a novel fluorogenic probe for peroxynitrite was proposed in this paper. The present method for the determination of peroxynitrite is simple, fast and sensitive. The response time of the proposed probe for peroxynitrite is less than 30 s, and it has a detection limit of 2.4× 10 − 8 mol l − 1 for peroxynitrite. Compared with the two fluorescent probes (DCFH and DHR-123) reported in the literature, the florescent probe reported here have some advantages: (i) longer excitation wavelength. DCFH and DHR-123 have the excitation wavelength of 502 and 500 nm [24], respectively, while the proposed probe has the longer wavelength of 556 nm. This is desirable for the probing of peroxynitrite formation in biological samples because of the low background fluorescence with the longer wavelength excitation and less cytotoxicity caused by longer UV excitation. (ii) Insensitive to pH change in a wider range. Because the rhodamine fluorophore does not have phenolic hydroxyl groups, the fluorescence of the reaction product of II with peroxynitrite would be independent of physiological pH change and can be applicable in a wider pH range. (iii) Higher stability. II demonstrates Table 1 The response of II (2.0×10−6 mol l−1) for a variety of potential interferences expressed as a percentage of the DF of peroxynitrite (7.5×10−7 mol l−1) in pH 7.4 of phosphate buffer Interference
Concentration (10−6 mol l−1)
Peroxynitrite (%)
Peroxynitrite NO− 2 NO− 3 NH2OH H2O2 Glucose Cystein Methionine Reduced glutathione (GSH) Ascorbic acid Epinephorine Cu2+
0.75 1000 1000 1000 100 1000 200 1000 200
100 (DF=122.4) 1.3 −0.3 1.1 0.2 1.4 3.0 −1.1 0.5
100 100 20
0.3 2.0 8.1
889
greater photostability than those of DCFH and DHR-123, which are extremely sensitive to light induced oxidation. Hence, we believe that the proposed probe can be applicable to study the peroxynitrite formation in biological systems.
Acknowledgements This work was supported by Natural Science Foundation of Fujian Province (D9920001) and National Education Committee of China.
References [1] S. Goldstein, G. Czapski, Free Radical Biol. Med. 19 (1995) 505. [2] R. Radi, J.S. Beckman, K.M. Bush, B.A. Freeman, J. Biol. Chem. 266 (1991) 4244. [3] R. Radi, J.S. Beckman, K.M. Bush, B.A. Freeman, Arch. Biochem. Biophys. 288 (1991) 481. [4] J.W. Reed, H.H. Ho, W.L. Jolly, J. Am. Chem. Soc. 96 (1974) 453. [5] N. Fukuyama, Y. Takebayashi, M. Hida, H. Ishida, K. Ichimori, H. Nakazawa, Free Radical Biol. Med. 22 (1997) 771. [6] C. Szabo, Shock 6 (1996) 79. [7] L. Thomson, M. Trujillo, R. Telleri, R. Radi, Arch. Biochem. Biophys. 319 (1995) 491. [8] R. Radi, T.P. Cosgrove, J.S. Beckman, B.A. Freeman, Biochem. J. 290 (1993) 51. [9] N.W. Kooy, J.A. Royall, Arch. Biochem. Biophys. 310 (1994) 352. [10] J. Xue, X. Ying, J. Chen, Y. Xian, L. Jin, Anal. Chem. 72 (2000) 5313. [11] S.B. Digerness, K.D. Harris, J.W. Kirklin, F. Urthaler, L. Vierera, J.S. Beckman, Free Radical Biol. Med. 27 (1999) 1386. [12] H. Possel, H. Noack, W. Augustin, G. Keihoff, G. Wolf, FEBS Lett. 416 (1997) 175. [13] S.L. Hempel, G.R. Buettner, Y.Q. O’Malley, D.A. Wessels, D.M. Flaherty, Free Radical Biol. Med. 27 (1999) 146. [14] N.W. Kooy, J.A. Royall, H. Ischiropoulos, J.S. Beckman, Free Radical Biol. Med. 16 (1994) 149. [15] N.W. Kooy, J.A. Royall, H. Ischiropoulos, Free Radical Res. 27 (1997) 245. [16] V. Dujols, F. Ford, A.W. Czarnik, J. Am. Chem. Soc. 119 (1997) 7386. [17] M.N. Hughes, H.G. Nicklin, J. Chem. Soc. A. (1968) 450. [18] J.W. Reed, H.H. Ho, W.L. Jolly, J. Am. Chem. Soc. 96 (1974) 1248.
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[19] J.R. Leis, M.E. Pena, A. Rios, J. Chem. Soc., Chem. Commun. (1993) 1298. [20] M.N. Hughes, H.G. Nicklin, J. Chem. Soc. A (1971) 164. [21] R. Radi, J.S. Beckman, K.M. Bush, B.A. Freeman, J. Biol. Chem. 266 (1991) 4244.
[22] J.W. Coddington, J.K. Hurst, S.V. Lymar, J. Am. Chem. Soc. 121 (1999) 2438. [23] S. Goldstein, G. Czapski, Inorg. Chem. 34 (1995) 4041. [24] R. Radi, G. Peluffo, M.N. Alvarez, M. Naviliat, A. Cayota, Free Radical Biol. Med. 30 (2001) 463.
Development of a novel rhodamine-type fluorescent probe to determine peroxynitrite Xiao-Feng Yang, Xiang-Qun Guo *, Yi-Bing Zhao Department of Chemistry and the Key Laboratory of Analytical Sciences of MOE, Xiamen Uni6ersity, Xiamen 361005, Fujian, PR China Received 13 December 2001; received in revised form 20 March 2002; accepted 22 March 2002
Abstract A novel method for the determination of peroxynitrite using rhodamine B hydrazide as a fluorogenic probe is described. The method is based on the oxidation of rhodamine B hydrazide, a colorless, non-fluorescent substance, by peroxynitrite to give rhodamine B-like fluorescence emission. The fluorescence increase is linearly related to the concentration of peroxynitrite in the range of 7.5 × 10 − 8 – 3.0×10 − 6 mol l − 1 with a detection limit of 2.4 × 10 − 8 mol l − 1 (3|). The optimal conditions for the detection of peroxynitrite were evaluated and the possible detection mechanism was also discussed in this paper. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Peroxynitrite; Rhodamine B hydrazide; Fluorimetry; Probe
1. Introduction Peroxynitrite, the product of the combination reaction between nitric oxide (NO) and superox ide radical (O− 2 ) [1], is a potent biological oxidant that promotes oxidative molecular and tissue damage [2,3]. Peroxynitrite formation and reactions are proposed to contribute to the pathogenesis of a series of diseases, including inflammatory processes, ischemia-reperfusion, septic shock and neurodegenerative disorders [4 – 6]. Study of the role of peroxynitrite needs real-time and sensitive probing of peroxynitrite in both pathological and * Corresponding author. Tel.: + 86-592-21-82442; fax: + 86-592-21-88054. E-mail address: [email protected] (X.-Q. Guo).
normal conditions in biological systems, which is indeed extremely difficult because of the low concentration, high activity and elusive nature of peroxynitrite. Peroxynitrite generation is usually measured by UV –visible spectrometry [7], chemiluminescence [8,9], electrochemistry [10], and immunohistochemistry [11]. Two fluorogenic probes, dihydrodichlorofluorescein (DCFH) and dihydrorhodamine-123 (DHR-123), have been widely employed to monitor peroxynitrite production in a variety of systems [12 –15]. The methods are based on the use of chemically reduced, nonfluorescent forms of highly fluorescent dyes such as fluorescein and rhodamine that are oxidized by peroxynitrite to the parent dye molecule, resulting in a dramatic increase in fluorescence intensity.
0039-9140/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 2 ) 0 0 1 2 0 - 0
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Thus, these ‘dihydro’ derivates can serve as fluorogenic probes for monitoring peroxynitrite production in biological systems. Rhodamine derivatives are prized for their great photostability, pH insensitivity over a broad range (low to neutral pH), high quantum yield in aqueous solution and to be excitable at long wavelength, and have been widely used for studying biological systems. In the present study, the characteristic of a non-fluorescent spiro form rhodamine B hydrazide was studied and the results showed that it has a sensitive response to proxynitrite. The reaction of peroxynitrite with rhodamine B hydrazide, a colorless, non-fluorescent compound results in a dramatic increase in fluorescence intensity of the reaction mixture. Based on this phenomenon, a novel fluorescent probe for peroxynitrite was developed. The objectives of the present study are to evaluate the ability of rhodamine B hydrazide as a probe for peroxynitrite and its potential use in biological systems. 2. Experimental
2.1. Chemicals Stock solution of rhodamine B hydrazide (II, 1.0 ×10 − 3 mol l − 1) was prepared by dissolving appropriate amount of II in 30% acetonitrile– water solution. Superoxide dismutase (SOD) (from bovine erythrocytes, 3000 U mg − 1) was obtained from Sigma. A dimethyl sulfoxide (DMSO, 1.0 mol l − 1) solution was prepared. A 1/15 mol l − 1 of KH2PO4 –Na2HPO4 buffer (pH 7.4) was also prepared.
All the reagents were of analytical-reagent grade, and doubly distilled water was used throughout.
2.2. Synthesis of rhodamine B hydrazide (II) A modification of the procedure of Dujols et al. [16] for the synthesis of II was employed. II was synthesized by a one-step reaction of rhodamine B (I) with hydrazine hydrate in methanol (Scheme 1). To a 0.4 g of rhodamine B (I) dissolved in 15 ml of methanol, an excessive hydrazine hydrate (0.5 ml) was added and then the reaction solution was refluxed till the pink color disappeared. After that, the cooled reaction solution was poured into distilled water and extracted with ethyl acetate (6× 25 ml). The combined extracts were dried with sodium sulfate anhydrous, filtered, and then evaporated. The solvent yielded 0.27 g (68%) of II. MS (CI) m/e 457.3, ([M+ H]+); M+, calculated 456.2. 1H NMR (CDCl3): l 7.93 (m, 1H, ArH), 7.45 (m, 2H, ArH), 7.11 (m, 1H, ArH), 6.46 (d, 2H, xanthene-H), 6.42 (d, 2H, xantheneH), 6.29 (dd, 2H, xanthene-H), 3.61 (s, 2H, NH2), 3.34 (q, 8H, NCH2CH3), 1.17 (t, 12H, NCH2CH3).
2.3. Synthesis of peroxynitrite Peroxynitrite was synthesized by the autoxidation of hydroxylamine in alkaline medium. The procedure was as follows: the mixture solution containing 0.01 mol l − 1 of hydroxylamine, 0.5 mol l − 1 of sodium hydroxide and 0.001 mol l − 1 of EDTA, was stirred vigorously in aerobic conditions for about 4–5 h, then some MnO2 powder
Scheme 1.
X.-F. Yang et al. / Talanta 57 (2002) 883–890
885
Scheme 2.
was added to the mixture solution to eliminate H2O2 generated in the reaction solution, after that the mixture was filtered and stored at − 18 °C. Peroxynitrite concentration was determined by UV spectrometry at 302 nm (m = 1670 l mol − 1 cm − 1) [17].
2.4. Apparatus The fluorescence spectra and relative fluorescence intensity were measured with a Shimadzu RF-5000 spectrofluorimeter (Kyoto, Japan) with a 10 mm quartz cuvette. The excitation and emission wavelength band-passes were both set at 5 nm. Absorption spectra were obtained on a Beckman DU-7400 Ultraviolet– Visible Spectrophotometer. All pH values were measured with a pH S-301 digital ion meter.
acid (ONOOH), to which NaOH is added immediately to generate the peroxynitrite anion (ONOO−) [18]. However, the elusive nature of ONOOH (t1/2, 1 s) makes the ‘timing’ of both processes (nitrosation and addition of NaOH) the key point for the success of the procedure. Therefore, this method often requires the use of a quenched-flow reactor. Another commonly used method is based on the reaction of H2O2 with alkyl nitrites in alkaline solutions [19]. This method is clean, simple and fast, but alkyl nitrite is not widely available. Hughes et al. had reported that the autoxidation of hydroxylamine in alkaline solutions led to the formation of peroxynitrite as the major product [20]. Hence, we synthesized peroxynitrite based on this reaction (Scheme 2). Fig. 1 shows the typical spectra of the system at
2.5. Procedure In a set of 10 ml-volumetric tubes containing pH 7.4 of phosphate buffer solution, 1.0 ml of II (2.0 ×10 − 5 mol l − 1) and different amount of peroxynitrite were added. The reaction solution was kept at room temperature for 2 min, and then the reaction solution was diluted to the mark with water. The fluorescence intensity of the solution was recorded at 574 nm with the excitation wavelength set at 556 nm.
3. Results and discussion
3.1. Preparation of peroxynitrite There are several methods reported for the synthesis of peroxynitrite. The commonly used method for the synthesis of peroxynitrite is based on the fast nitrosation of H2O2 in acid medium to yield the solution of the unstable peroxynitrous
Fig. 1. Typical reaction spectrum showing the formation of peroxynitrite from the autoxidation of hydroxylamine in alkaline solution. Hydroxylamine, 0.01 mol l − 1; NaOH, 0.5 mol l − 1; EDTA, 0.001 mol l − 1. The reaction was carried out at aerobic condition at room temperature (20 °C), with vigorous stirring. The spectrum was recorded by transferring 2.0 ml of reaction mixture to a 10-ml volumetric tube at different reaction time and diluting to the mark with water. t: (a) 5 min; (b) 20 min; (c) 35 min; (d) 55 min; (e) 75 min; (f) 95 min; (g) 115 min; (h) 135 min; (i) 155 min.
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excitation nor fluorescence emission spectra. However, a mixture of II with peroxynitrite shows apparent spectral characteristics with excitation maximum at 556 nm and fluorescence emission maximum at 574 nm.
3.3. Optimization of the general procedure
Fig. 2. Fluorescence excitation and emission spectra of the system. a, a%, II (2.0 ×10 − 6 mol l − 1); b, b%, II (2.0×10 − 6 mol l − 1)+ peroxynitrite (7.5 × 10 − 7 mol l − 1); c, c%, rhodamine B (2.5 ×10 − 8 mol l − 1). The reaction of peroxynitrite with II was carried out in phosphate buffer (pH 7.4) at room temperature (20 °C) for 2 min. All the spectra were recorded at pH 7.4 of phosphate buffer.
different stages of reaction. As the reaction proceeds, a new band centered on 300– 302 nm, corresponding to ONOO− is formed. EDTA is added as the metal-ion sequestering to the reaction solution to preclude the decomposition of peroxynitrite. The peroxynitrite concentration is estimated from absorbance at 302 nm, nitrate and peroxide scarcely shows absorbance in this region (m= 7 and 40 l mol − 1 cm − 1, respectively) [20]. Moreover, the coexisting species, such as hydrox− ylamine, NO− 3 , NO2 , and H2O2 show no response to the fluorescence increase of the solution of II (see Section 3.6). The present method for the preparation of peroxynitrite is easy to operate, needs no special equipment and only inexpensive and easy available reagents are used.
3.3.1. Effect of reaction time The kinetic characteristics of the proposed detection system were studied. Upon the addition of peroxynitrite to the solution of II in phosphate buffer, the fluorescence intensity of the detection system was recorded as a function of reaction time (as shown in Fig. 3). From Fig. 3, one may see the fluorescence intensity of the detection system increased dramatically in a few seconds and leveled off as the reaction went on, while the fluorescence background of the detection system in the absence of peroxynitrite remained unchanged at the same time. The fluorescence intensity of the detection system reached its maximum value for about 1 min, after that the fluorescence intensity of the detection system remained almost constant. To obtain a high sensitivity and reproducible results, a 2-min reaction time was selected in the following experiment.
3.2. Spectra characteristics II is a colorless, non-fluorescent substance. When peroxynitrite was introduced to the solution of II, a fluorescence emission similar to that of rhodamine B was observed. Fig. 2 shows the fluorescence excitation and emission spectra of II and the mixture of II with peroxynitrite in phosphate solution (pH 7.4). It can be seen that II shows no obvious spectra characteristics, neither
Fig. 3. Kinetic behavior of the present system. (a) II; (b) II+ peroxynitrite (7.5 × 10 − 7 mol l − 1); (c) II + peroxynitrite (1.5 ×10 − 6 mol l − 1). II, 2.0 × 10 − 6 mol l − 1. The fluorescence development and measurement were both carried out in pH 7.4 of phosphate buffer solution.
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concentration of II was up to 1.2× 10 − 6 mol l − 1, therefore, 2.0×10 − 6 mol l − 1 of II was recommended for the subsequent experiment.
Fig. 4. Effect of pH on the fluorescence development reaction of the system. The reaction of II (2.0× 10 − 6 mol l − 1) with peroxynitrite (7.5 ×10 − 7 mol l − 1) was carried out in phosphate buffer with different pH value for 2 min, then pH of the system was adjusted to 7.4 and the fluorescence intensity was recorded.
3.3.2. Effect of pH The effect of pH on the fluorogenic reaction was studied in the range of 5.0– 9.1 in phosphate buffer solution, and the results are shown in Fig. 4. It can be seen that fluorescence increment (DF) of the detection system was increased with pH up to 6.8, remained almost the same in the range of 6.8 – 7.7, and decreased when pH value was above 7.7. Hence, pH 7.4 of phosphate buffer was chosen for fluorogenic reaction in the following study. It is reported that peroxynitrite has a pKa of 6.8 [21], so at pH 9.0, about 99.4% of peroxynitrite is present in the form of ONOO−, while at pH 5.0, about 98.4% of peroxynitrite is present as the conjugate acid, ONOOH. From Fig. 4, it can be seen that the fluorescence increase of the system be observed in a significant degree at pH 5.0 and at 9.0, respectively, indicating that both ONOOH and ONOO− can oxidize II to yield a highly fluorescent product, which is consistent with the oxidizing nature of both species. 3.3.3. Effect of the concentration of II The effect of the concentration of II on DF of the system was studied and the results were shown in Fig. 5. From Fig. 5, it can be seen that DF of the detection system was increased with increasing the concentration of II from 4.0× 10 − 7 to 1.2× 10 − 6 mol l − 1 and remained constant when the
3.3.4. Stability of II The stability of II was studied at different pH values and the experiments showed the fluorescence background of the system varied with pH. In strongly acid media (0.1 mol l − 1 sulfuric acid), the fluorescence background was unstable and increased with time, presumably because II was partly decomposed in strongly acid media and hence a high fluorescence background was recorded, while the fluorescence background of the proposed system was low and stable when the pH of the detection system was ]3. To maintain its stability, II should be kept in neutral or near neutral pH solution, and the water solution of II was able to stand at room temperature for 3 weeks without apparent increase of fluorescence background. (Further experiment on the stability of II with time was not carried out.) 3.4. Possible detection mechanism The oxidation of the various substrates by peroxynitrite (OONO−/ONOOH) can take place via multiple pathways: (i) peroxynitrite may directly
Fig. 5. Effect of the concentration of II on the fluorescence increment (DF) of the system. II (different concentrations) + peroxynitrite (7.5 ×10 − 7 mol l − 1) +phosphate buffer (pH 7.4). The reaction mixture was carried out at room temperature (20 °C) for 2 min, and then the fluorescence intensity was recorded.
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Scheme 3.
oxidize the substrates. (ii) Peroxynitrite may decompose firstly into highly reactive species (OH, NO2), which subsequently oxidizes the substrate or hydroxylates and nitrates aromatic compound. In the present study, the possible mechanism of the oxidation of II by peroxynitrite is studied. Firstly, DMSO (0.1 mol l − 1), a specific scavenger for OH, was introduced to the reaction mixture before the addition of peroxynitrite, and we found the DF of the reaction mixture was almost unchanged, indicating that OH does not contribute to the fluorescence increase of the system. Second, SOD (3 U ml − 1), a specific scavenger for O− 2 , was added to the reaction system, and the fluorescence of the system showed no changes compared with that in the absence of SOD, proving that does not mediate the fluorescence increase of O− 2 the system. The above experiments suggest that the oxidation of II might arise from the peroxynitrite itself, but not its decomposed reactive species. This can be explained that the reaction rate of peroxynitrate with II (k4) was faster than the self-decomposition rate of peroxynitrite via the pathway of 2 and 3 described in Scheme 3 (k2, k3 are 0.017 and 0.8 s − 1, respectively [22]. k4 is still unknown, but according to the literature, which reported the reaction rate of direct oxidation of some compounds by peroxynitrite were 104 l mol − 1 s − 1 [23], we assume that the value of k4 is 104 l mol − 1 s − 1). Third, comparison of the excitation and emission fluorescence spectra of the reaction system with that of authentic rhodamine B shows that they are identical, both having excitation maximum at 556 nm and emission maximum at 574 nm (shown in Fig. 2), indicating that the fluorescent product generated in the reaction
mixture might be rhodamine B. Based on the above experimental results, a possible mechanism of the proposed method is given by Scheme 3.
3.5. Limit of detection, linear concentration range and precision Under the selected conditions given above, the fluorescence increment shows a linear relationship with the concentration of peroxynitrite in the range of 7.5×10 − 8 –3.0×10 − 6 mol l − 1 (r= 0.9991, n= 8). The detection limit is 2.4× 10 − 8 mol l − 1 (3|). The relative standard deviation (n= 7) is 4.1% for 7.5× 10 − 7 mol l − 1 of peroxynitrite.
3.6. Interference study The interferences of the proposed probe for peroxynitrite were studied. A variety of interfering agents were added to the solution of II in pH 7.4 of phosphate buffer, and the response of II to these compounds are listed in Table 1. Interestingly, it was found that as high as 10 − 5 mol l − 1 of Cu2 + did not produce significant fluorescence increment of the detection system described in this paper, which is not contradicted with the fact described by Dujols et al. [16], because the selective response of II to Cu2 + must be carried out in 20% acetonitrile–water solution. If 20% of acetonitrile was added to the solution of II containing Cu2 + , a simultaneous fluorescence increment was observed. Hence, the proposed probe for peroxynitrite without acetonitrile could successfully avoid the interference from Cu2 + .
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4. Conclusion In summary, a novel fluorogenic probe for peroxynitrite was proposed in this paper. The present method for the determination of peroxynitrite is simple, fast and sensitive. The response time of the proposed probe for peroxynitrite is less than 30 s, and it has a detection limit of 2.4× 10 − 8 mol l − 1 for peroxynitrite. Compared with the two fluorescent probes (DCFH and DHR-123) reported in the literature, the florescent probe reported here have some advantages: (i) longer excitation wavelength. DCFH and DHR-123 have the excitation wavelength of 502 and 500 nm [24], respectively, while the proposed probe has the longer wavelength of 556 nm. This is desirable for the probing of peroxynitrite formation in biological samples because of the low background fluorescence with the longer wavelength excitation and less cytotoxicity caused by longer UV excitation. (ii) Insensitive to pH change in a wider range. Because the rhodamine fluorophore does not have phenolic hydroxyl groups, the fluorescence of the reaction product of II with peroxynitrite would be independent of physiological pH change and can be applicable in a wider pH range. (iii) Higher stability. II demonstrates Table 1 The response of II (2.0×10−6 mol l−1) for a variety of potential interferences expressed as a percentage of the DF of peroxynitrite (7.5×10−7 mol l−1) in pH 7.4 of phosphate buffer Interference
Concentration (10−6 mol l−1)
Peroxynitrite (%)
Peroxynitrite NO− 2 NO− 3 NH2OH H2O2 Glucose Cystein Methionine Reduced glutathione (GSH) Ascorbic acid Epinephorine Cu2+
0.75 1000 1000 1000 100 1000 200 1000 200
100 (DF=122.4) 1.3 −0.3 1.1 0.2 1.4 3.0 −1.1 0.5
100 100 20
0.3 2.0 8.1
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greater photostability than those of DCFH and DHR-123, which are extremely sensitive to light induced oxidation. Hence, we believe that the proposed probe can be applicable to study the peroxynitrite formation in biological systems.
Acknowledgements This work was supported by Natural Science Foundation of Fujian Province (D9920001) and National Education Committee of China.
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