Simultaneous Determination of Superoxide and Hydrogen Peroxide in

Anal. Chem. 2009, 81, 2193–2198

Simultaneous Determination of Superoxide and Hydrogen Peroxide in Macrophage RAW 264.7 Cell Extracts by Microchip Electrophoresis with Laser-Induced Fluorescence Detection Hongmin Li, Qingling Li, Xu Wang, Kehua Xu, Zhenzhen Chen, Xiaocong Gong, Xin Liu, Lili Tong, and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan 250014, People’s Republic of China A method for the first time to simultaneously determine superoxide and hydrogen peroxide in macrophage RAW 264.7 cell extracts by microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF) was developed. 2-Chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) and bis(p-methylbenzenesulfonyl) dichlorofluorescein (FS), two probes that can be specifically derivatized by superoxide and hydrogen peroxide, respectively, were synthesized and used. Parameters influencing the derivatization and on-chip separation were optimized. With the use of a HEPES (20 mM, pH 7.4) running buffer, a 50 mm long separation channel, and a separation voltage of 1800 V, baseline separation was achieved within 48 s for the two derivatization products, DBZTCoxide (DBO) and 2,7-dichlorofluorescein (DCF). The linearity ranges of the method were 0.08-5.0 and 0.02-5.0 µM with detection limits (signal-to-noise ratio ) 3) of 10 nM (1.36 amol) and 5.6 nM (0.76 amol) for superoxide and hydrogen peroxide, respectively. The relative standard deviations (RSDs) of migration time and peak area were less than 2.0% and 5.0%, respectively. The recoveries of the cell extract samples spiked with 1.0 µM standard solutions were 96.1% and 93.0% for superoxide and hydrogen peroxide, respectively. With the use of this method, superoxide and hydrogen peroxide in phorbol myristate acetate (PMA)-stimulated macrophage RAW 264.7 cell extracts were found to be 0.78 and 1.14 µM, respectively. The method has paved a way for simultaneously determining two or more reactive oxygen species (ROS) in a biological system with high resolution. Superoxide anion, the first one-electron reduction product of oxygen metabolism, implicates biological and pathophysiological roles in two aspects. First, it serves as a precursor for other reactive oxygen species (ROS), such as hydrogen peroxide, peroxynitrite, and hydroxyl radical, which can cause oxidative damage to lipids, proteins, and DNA. Second, it can inactivate NO, thereby causing endothelial dysfunction. Its concomitant molec* To whom correspondence should be addressed. Fax: 86-531-86180017. E-mail: [email protected]. 10.1021/ac801777c CCC: $40.75  2009 American Chemical Society Published on Web 02/10/2009

ular, hydrogen peroxide, receives the same concern because it acts as an endothelium-derived hyperpolarizing factor (EDHF),1-3 or functions as a second messenger in cell apoptosis,4 proliferation,5 and differentiation6 due to its cell-permeable, strong oxidizing, and far-reaching effect. Furthermore, these two ROS both participate in the physiological processes in a concentrationdependent manner. At low concentrations, they play an important role as regulatory mediators in signaling processes, whereas at high concentrations, they can damage major cellular constituents and are hazardous for living organisms. In biological milieu, superoxide and hydrogen peroxide always coexist. Once superoxide is produced, it will spontaneously or enzymatically dismute into hydrogen peroxide. In some cases, they play the same roles in pathophysiological conditions. But in most cases, they perform differently because of their specific chemical reactivity, such as in calcium regulation,7 mitogenactivated protein kinases activation,8 T-cell receptor stimulation,9 apoptosis, and proliferation of vascular smooth muscle cells.10 Therefore, it is very desirable to quantitatively measure superoxide as well as hydrogen peroxide in biological systems for the better understanding of their functions in physiological processes and many diseases. But it is not an easy task for the simultaneous detection of these two ROS due to their low homeostasis concentrations, high reactivity, and fast interconversion. Currently, the determination methods for superoxide or hydrogen peroxide mainly include electron spin resonance (ESR),11 chemiluminescence,12 and electrochemical detection,13 (1) Matoba, T.; Shimokawa, H. J. Pharm. Sci. 2003, 92, 1–6. (2) Shimokawa, H.; Matoba, T. Pharm. Res. 2004, 49, 543–549. (3) Matoba, T.; Shimokawa, H.; Nakashima, M.; Hirakawa, Y.; Mukai, Y.; Hirano, K.; Kanaidc, H.; Takeshita, A. J. Clin. Invest. 2000, 106, 1521– 1531. (4) Sauer, H.; Wartenberg, M.; Hescheler, J. Cell. Physiol. Biochem. 2001, 11, 173–186. (5) Turrens, J. F. J. Physiol. 2003, 552, 335–344. (6) Liu, H.; Colavitti, R.; Rovira, L. L.; Finkel, T. Circ. Res. 2005, 97, 967–974. (7) Tabet, F.; Savoia, C.; Schiffrin, E. L.; Touyz, R. M. J. Cardiovasc. Pharmacol. 2004, 44, 200–208. (8) Baas, A. S.; Berk, B. C. Circ. Res. 1995, 77, 29–36. (9) Devadas, S.; Zaritskaya, L.; Rhee, S. G.; Oberley, L.; Williams, M. S. J. Exp. Med. 2002, 195, 59–70. (10) Li, P. F.; Harsdorf, R. V. Circulation 1997, 96, 3602–3609. (11) Borbat, P. P.; Costa-Fihlo, A. J.; Earle, K. A.; Moscicki, J. K.; Freed, J. H. Science 2001, 291, 266–269.

Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

2193

etc. But all the aforementioned methods show unavoidable disadvantages. ESR is lack of spin trap specificity. Chemiluminescence often generates artifactual superoxide. Electrochemical detection is limited to the electrode area and its positioning. Moreover, the simultaneous detection of superoxide and hydrogen peroxide in biological samples by these methods is not realized yet. In the past decade, although different methods have been adopted to assess the generation of superoxide and hydrogen peroxide in parallel,14-16 excessive time and effort are normally included. Beside this, the most intractable aspect is the different lifetime and reciprocal conversion of these two ROS, resulting in their poorly simultaneous profile in a biological sample. The Lvovich group13 fabricated a four-electrode amperometric sensor to study the superoxide/hydrogen peroxide production ratio in a xanthine/xanthine oxidase (XA/XO) model, but it was not applied to biological samples. Therefore, it was urgent and would be ideal to develop a method that can separate and detect superoxide and hydrogen peroxide simultaneously. In comparison with conventional analytical methods, microfluidic chips or laboratory on a chip have exhibited advantages in terms of lower sample consumption, better portability, easier integration, and less analysis time.17-19 It was reported that with the most compliant fluorophores, microchip electrophoresis coupled with laser-induced fluorescence (MCE-LIF) can detect the species of interest in the samples even at the femtomolar level.20 Although fluorescence detection of ROS on microchip was realized by using rhodamine derivatives21-24 and hydroethidine25 the simultaneous recognition of two ROS was not realized because of the bulk response of rhodamine derivatives to total ROS and the mutual interference of oxided products of hydroethidine.26-28 To resolve these problems, the fluorogenic probes that are capable of detecting single ROS have to be designed and synthesized. Recently, 2-chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) synthesized by our group showed a highly selective response to superoxide.29 An amount of 500-fold hydrogen peroxide does not interfere, providing a better way to monitor superoxide in vitro. In addition, a fluorescent probe of bis(p-methylbenzenesulfonyl) (12) Faulkner, K.; Fridovich, I. Free Radical Biol. Med. 1993, 15, 447–451. (13) Lvovich, V.; Scheeline, A. Anal. Chem. 1997, 69, 454–462. (14) Carter, W. O.; Narayanan, P. K.; Robinson, J. P. J. Leukocyte Biol. 1994, 55, 253–258. (15) Narayanan, P. K.; Goodwin, E. H.; Lehnert, B. E. Cancer Res. 1997, 57, 3963–3971. (16) Han, D.; Canali, R.; Rettori, D.; Kaplowitz, N. Mol. Pharmacol. 2003, 64, 1136–1144. (17) Vilkner, T.; Janasek, D.; Manz, A. Anal. Chem. 2004, 76, 3373–3386. (18) Dittrich, P. S.; Tachikawa, K.; Manz, A. Anal. Chem. 2006, 78, 3887–3907. (19) Jacobson, S. C.; Koutny, L. B.; Hergenroeder, R.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472–3476. (20) Fister, J. C.; Jacobson, S. C.; Davis, L. M.; Ramsey, J. M. Anal. Chem. 1998, 70, 431–437. (21) Qin, J. H.; Ye, N. N.; Yu, L. F.; Liu, D. Y.; Fung, Y. S.; Wang, W.; Ma, X. J.; Lin, B. C. Electrophoresis 2005, 26, 1155–1162. (22) Ling, Y. Y.; Yin, X. F.; Fang, Z. L. Electrophoresis 2005, 26, 4759–4766. (23) Gao, N.; Li, L.; Jin, W. R. Electrophoresis 2007, 28, 3966–3975. (24) Sun, Y.; Yin, X. F.; Ling, Y. Y.; Fang, Z. L. Anal. Biochem. 2005, 382, 1472– 1476. (25) Zhu, L. L., Lu, M., Yin, X. F. Talanta 2008, 75, 1227-1233. (26) Huang, A. N.; Xiao, H.; Samii, J. M.; Vita, J. A.; Keaney, J. F. Am. J. Physiol. 2001, 281, 719–725. (27) Kuzkaya, N.; Weissmann, N.; Harrison, D. G.; Dikalov, S. J. Biol. Chem. 2003, 278, 22546–22554. (28) Zhao, H. T.; Kalivendi, S.; Zhang, H.; Joseph, J.; Nithipatikom, K.; VasquezVivar, J.; Kalyanaraman, B. Free Radical Biol. Med. 2003, 34, 1359–1368. (29) Gao, J. J.; Xu, K. H.; Tang, B. FEBS Lett. 2007, 274, 1725–1733.

2194

Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

dichlorofluorescein (FS) for hydrogen peroxide relying on the deprotection mechanism was synthesized in house, allowing the highly specific and peroxidase-independent detection of hydrogen peroxide in the complicated biological matrix. Moreover, the fluorescence properties of the derivative products, namely, DBO (λex/λem ) 485/559 nm) and DCF (λex/λem ) 491/525 nm), accommodate an argon ion laser detector very well and allow their efficient excitation at 473 nm. On the basis of this, we introduced the XA/XO system, a standard procedure for generating superoxide and hydrogen peroxide, as a cell-free model to characterize the specificity of the probes. In addition, the determination of superoxide and hydrogen peroxide was further confirmed by applying the proposed method to the phorbol myristate acetate (PMA)stimulated macrophage cells. It was found that, in the cellular extracts, the baseline separation of DBO and DCF was accomplished within 48 s, showing rapid detection ability. The sensitivity, accuracy, and reproducibility of the method were also satisfied. All of these make the developed method a fast and reliable approach for ROS detection in cell system and can be well extended to other biological systems. EXPERIMENTAL SECTION Reagents. All reagents were of analytical degrade and used without further purification unless otherwise noted. 4-(2-Hydroxyerhyl)piperazine-1-erhanesulfonic acid (HEPES), superoxide dismutase (SOD), catalase (CAT), XA, XO, dimethyl sulfoxide (DMSO), and PMA were from Sigma (American). DBZTC and FS were synthesized in house, and their stock solutions (1 mM) were prepared by dissolving appropriate amounts of DBZTC and FS in DMSO. For further using, the working solutions of DBZTC and FS were prepared by diluting the stock solutions into 6.00 × 10-4 and 1.20 × 10-3 M, respectively. The preparation and purification of DBZTC-oxide (DBO) followed the procedure from ref 29. Its stock solution was prepared by dissolving DBO in DMSO with concentration of 1.00 × 10-4 M. 2,7-Dichlorofluorescein (DCF) was from Alexi (American). Its stock solution (1.00 × 10-4 M) was prepared by dissolving an appropriate amount of DCF in DMSO. The XA solution (1.00 mM) was prepared by dissolving an appropriate amount of XA in 1.00 × 10-2 M NaOH. The stock solution of XO (1.00 U mL-1) was prepared by dissolving the appropriate amount of XO in a solution of 2.30 M (NH4)2SO4 and 1.00 × 10-2 M sodium salicylate buffer and was stored at 2-8 °C. The buffer solutions were filtered through a 0.22 µm membrane filter before introduction into the chip. Borate, phosphate, and HEPES buffers were prepared by the addition of appropriate amounts of hydrochloric acid or sodium hydroxide to a desired pH. Deionized water from a Milli-Q system was used throughout. Synthesis of FS and Its Characterization. The procedures of FS synthesis and its characterization by 1H NMR, FT-IR, and elemental analysis are provided in Supporting Information. Cell-Free Assays. ROS were generated by a XA/XO system in HEPES (20 mM), pH 7.4, at 37 °C. The reaction mixture contained DBZTC (10 µM), FS (20 µM), XA (1 × 10-6 M), and XO (1 × 10-6 U mL-1). In independent experiments, SOD (1.0 U) and CAT (1.0 U) were added as superoxide and hydrogen peroxide scavengers, respectively.

Scheme 1. Derivatization of the Fluorescent Probes by ROS

Figure 1. Schematic diagrams of channels design of the microfluidic chip (A) and the chip-based CE setup (B). S, sample reservoir; SW, sample waste reservoir; B, buffer reservoir; BW, buffer waste reservoir; IEED, intelligent eight-channel high-voltage electric device; LIF-D, laser-induced fluorescence detector.

RAW 264.7 Macrophages Cultures. RAW 264.7 macrophages were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 1.0% penicillin, and 1.0% streptomycin in a 5% CO2/95% air incubator (MCO-15AC, Sanyo). The concentration was counted to be 1 × 106 cells mL-1. A set of cells was stimulated with PMA (2.0 ng/mL) at 37 °C for 12 h. Then they were washed with DMEM, and 2.5 mL DMEM was loaded. After that, DBZTC (0.06 mM, 0.50 mL) and FS (0.12 mM, 0.50 mL) were added. The solutions were incubated for 30 min at 37 °C for further experiment. RAW 264.7 Macrophage Extracts. After incubation for 30 min at 37 °C, the cells were harvested by centrifugation and washed twice with 0.10 M HEPES solution. After that, they were suspended again in 0.02 M HEPES with the same volume as they had been grown and disrupted for 10 min in a VC 130PB ultrasonic disintegrater (Sonics & Materials Inc.). During sonic disruption, the temperature was maintained below 4 °C with circulating ice water. The broken cell suspension was centrifuged at 8000 rpm for 15 min, and the pellet was discarded. The PMA-stimulated cells suspension was divided into six parts, and standard DBO and DCF solutions of 1.0 µM were added into three parts in turn for the recovery studies. The supernatants were immediately analyzed or kept at -80 °C for up to 2 weeks. Microchip Fabrication. A glass microchip with simple cross design was fabricated in Dalian Institute of Chemical Physics, Chinese Academy of Sciences (Dalian, China). The channels were etched onto one glass plate to which a top plate was bonded. The layout and dimensions of the electrophoretic microchip are depicted in Figure 1A. The channels were 60 µm wide and 20 µm deep, and the laser detection point lies 10 mm downstream from the sample reservoir cross the separation channels. The glass microchip was mounted onto a cassette to provide a fluidic interface and to make electrical connections between the buffer reservoirs and the high-voltage power supply.

MCE-LIF Detection. A homemade MCE-LIF system with an intelligent eight-channel high-voltage electric device (IEED) and confocal optical structure is displayed in Figure 1B.30 A semiconductor double-pumped solid-state laser (SDPSS, MBL-20, Changchun Xinchanye Guangdianjishu Co. Ltd., Changchun, China) with 473 nm was used as a light source. A narrow band filter with the wavelength of 525 ± 10 nm (Omega Optical, Brattleboro, VT) was used. A photomultiplier (PMT) (CR131, Hamamatsu, Japan) was used as the fluorescence detector. The sampling frequency of CT22 data acquisition card (Shanghai Qianpu Shuju Co. Ltd., Shanghai, China) was 20 Hz. Electrophoresis Procedures. The microchip was cleaned sequentially with 1 M sodium hydroxide (15 min) and distilled water (10 min). Then it was flushed with running buffer for 10 min. Sample injection was carried out using a pinch mode. Sample injection was performed for 30 s by applying +300 V potential across the sample reservoir (S) and sample waste reservoir (SW). Voltages of +120 V and +140 V were applied to the buffer reservoir (B) and buffer waste reservoir (BW) during the injection step. Separation was immediately carried out following the injection by applying +1800 V to buffer reservoir (B) and +1300 V to the sample reservoir (S) and sample waste reservoir (SW). Safety Considerations. The high-voltage power supply should be handled with extreme care to avoid electrical shock. PMA is highly toxic and should be handled in a fume hood. Skin and eye contact and accidental inhalation or ingestion should be avoided. RESULTS AND DISCUSSION Optimization of Derivatization of DBZTC and FS. The derivatization mechanisms of DBZTC and FS by ROS are given in Scheme 1. As the derivatization conditions of DBZTC has been investigated in our previous work,29 we focused on the derivatization of FS. To find out the optimum reaction conditions for the analysis of hydrogen peroxide, the effects of buffer solution, incubation time, and concentration of fluorescent probe were investigated. HEPES buffer solution was selected because it is a suitable buffer for mammalian cell incubation and can improve planting efficiency and breed ability of cells. FS exhibited maximum fluorescence response when it was incubated at 37 °C for 30 min in the presence of 20 mM HEPES (pH 7.4). Moreover, the concentration of FS determined the capturing efficiency of hydrogen peroxide, as well as the preciseness and sensitivity of (30) Li, Q. L.; Tang, B.; Tian, H. X. Chinese Patent No. 200510104343.3, 2005.

Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

2195

Figure 2. Effect of buffer pH on the fluorescence intensity of 1.0 µM DBO and 1.0 µM DCF in 30 mM HEPES buffer. Separation conditions: injection time, 30 s; separation voltage, 300 V/cm; effective separation distance, 10 mm. Each point represents an average of experiments repeated for three times.

the method. It was found that the fluorescence intensity increased with increasing FS concentration up to 20 µM, after which fluorescence quenching was induced because of self-absorption. Therefore, 20 µM of FS was selected. Specificity of DBZTC and FS. The specificity of DBZTC to superoxide was studied in our previous work.31 Results showed that 500-fold hydrogen peroxide will not interfere. Specificity of FS to hydrogen peroxide was investigated in the presence of several kinds of potential interfering substances, including ROS, reductants, glutathione (GSH), and esterase. The solution of FS (1.0 µM, in DMSO-HEPES buffer) was first incubated with hydrogen peroxide (1.0 µM) at 37 °C for 30 min. Then its fluorescence response (λex/ em ) 493/525 nm) was compared to those reacted with O2-•, •OH, NaOCl (1.0 µM), t-BuOOH (1.0 µM), GSH (1.0 µM), 3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene (NOC-5) (1.0 µM), esterase (1.0 µM), 3-morpholinosydnonimine (SIN-1) (1.0 µM), ascorbic acid (1.0 µM), and 1,4-hydroquinone (1.0 µM). The results showed that FS gave the highest signal with hydrogen peroxide, and little response was observed with ascorbic acid, glutathione, 1,4-hydroquinone, esterase, and other ROS, especially to •OH. Therefore, FS can be used as probe for hydrogen peroxide in the subsequent experiment. Optimization of MCE Separation. The composition, pH, and concentration of running buffer have a dramatic effect upon the MCE separation. It was reported that DCF exhibits good peak shape using the high-pH borate buffer,31,32 but this was not suitable for DBO which shows poor signal response in the same buffer. A HEPES buffer offers higher resolution and more sensitive response for DBO compared to phosphate and borate buffer. To obtain a compromise, HEPES buffer was selected. In addition, the effect of buffer pH was examined, which is shown in Figure 2. As can be seen from Figure 2, the fluorescence intensity of DCF was pH-dependent33 and its fluorescence increased with increasing pH in the range of 7.0-8.2. As to DBO, it exhibited the biggest fluorescence response in pH 7.4. Besides pH, the ionic strength of running buffer has to be optimized because of its apparent effect on migration time and signal-to-noise level. When (31) Kim, M. S.; Cho, S. I.; Lee, K. N.; Kim, Y. K. Sens. Actuators, B 2005, 107, 818–824. (32) Jeong, Y. W.; Choi, K. W.; Kang, M. K.; Chun, K. J.; Chung, D. S. Sens. Actuators, B 2005, 104, 269–275. (33) Desbene, P. L.; Morin, C. J.; Monvernay, A. M. D.; Groult, R. S. J. Chromatogr., A 1995, 689, 135–148.

2196

Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

Figure 3. Effect of the electric field on the migration time (A) and plate number N (B) of a solution containing 1.0 µM DBO and 1.0 µM DCF in 20 mM HEPES buffer. Separation conditions: injection time, 30 s; effective separation distance, 10 mm. Each point represents an average of experiments repeated for three times.

Figure 4. Typical electropherogram of standard solutions of 1.0 µM DBO and 1.0 µM DCF in 20 mM HEPES buffer (pH 7.4). Separation conditions: injection time, 30 s; separation voltage, 360 V/cm; effective separation distance, 10 mm; RFU (mV), relative fluorescence units.

the buffer concentration increases, both the migration time and signal-to-noise of DBO and DCF increase. Therefore, a concentration of 20 mM as a compromise between migration time and signal-to-noise was selected for further experiments. The effect of the electric field upon migration time and separation efficiency is shown in Figure 3A. As expected, increasing the separation potential from 200 to 360 V/cm dramatically decreases the migration time for both DBO and DCF, which decreases from 60 to 32 s and 70 to 40 s, respectively. The effect of separation voltage upon the theoretical plate numbers (N) for DBO and DCF is shown in Figure 3B. As can be seen, the maximum N values are obtained at a electric field of 360 V/cm. The decreased separation efficiency at high field strength can be attributed to Joule heating. Linearity, Reproducibility, and Detection Limits. A representative electropherogram of DBO and DCF is shown in Figure

Table 1. Linearity, Reproducibilities, and Limits of Detection (LODs) for the Two ROS (n ) 10) ROS

regression equation

coefficient of correlation

RSD (%) (migration time)

RSD (%) (peak area)

LOD (nM)

superoxide hydrogen peroxide

Y ) (1.2 ± 0.26) + (0.57 ± 0.02)Xa,b Y ) (0.33 ± 0.12) + (1.1 ± 0.050)X

0.9987 0.9939

0.80 1.2

4.6 4.0

10 5.6

a

Y: the peak area count. b X: the concentration in micromoles per liter.

Figure 5. Electropherograms of DBO and DCF formed by the xanthine/xanthine oxidase reaction. The reaction mixture contained 10 µM DBZTC, 20 µM FS, 1.0 µM xanthine, and 1.0 µM xanthine oxidase in HEPES buffer.

4, demonstrating the excellent separation of these two adducts under the optimized conditions. The migration speed of DBO was faster than that of DCF. This elution order can be explained on the basis of the molecular structure and charge-to-mass ratio. In the weak alkaline solution of HEPES with pH 7.4, DBO exists as neutral form, resulting in faster migration than DCF, which was negatively charged and migrated against the electro-osmotic flow (EOF). The linearity, reproducibility, and detection limits for the determination of DBO and DCF are listed in Table 1. A linearity of nearly 2 orders of magnitude was obtained for DBO and DCF with range of 0.08-5.0 µM for superoxide and 0.02-5.0 µM for hydrogen peroxide, respectively, Reproducibility in terms of relative standard deviations (RSDs) for migration time and relative peak area was calculated by using 1.0 µM DBO and DCF solutions (n ) 10). The RSDs of migration time for DBO and DCF were 0.80% and 1.20%, respectively, whereas those of peak area were 4.6% and 4.0%, respectively. The detection limits calculated from 3-fold signal-to-noise for superoxide and hydrogen peroxide were 10 and 5.6 nM, corresponding to 1.36 and 0.76 amol in mass limit (considering a repeatable injection volume of 136 pL with simple cross design). Cell-Free Assay. It has been reported that XA can simultaneously generate superoxide and hydrogen peroxide via one- and two-electron pathways under the catalysis of XO.34 Two peaks corresponding superoxide and hydrogen peroxide are observed after 10 min of incubation, and the peak height increases with time (Figure 5). After 30 min of incubation, the peaks become stable. DBO and DCF peaks in the system were identified by scavenging method. It is well-known that SOD and CAT are the specific digest enzymes to superoxide and hydrogen peroxide, respectively. The results (Figure 6) indicate that the two peaks were assigned to superoxide and hydrogen peroxide, respectively. (34) Benov, L.; Sztejnberg, L.; Fridovich, I. Free Radical Biol. Med. 1998, 25, 826–831.

Figure 6. Electropherograms of DBO and DCF formed by the xanthine/xanthine oxidase reaction in the presence of 1.0 U/mL SOD (B) and 1.0 U/mL CAT (C). The reaction mixture contained 10 µM DBZTC, 20 µM FS, 1.0 µM xanthine, and 1.0 µM xanthine oxidase in HEPES buffer.

Figure 7. Electropherogram of DBO and DCF in cell extracts. Other conditions were the same as in Figure 4.

In the process of scavenging, SOD dismuted superoxide into hydrogen peroxide, which resulted in a higher DCF peak and disappearance of the DBO peak. In the presence of CAT, the peak of DCF disappeared because of dismutation of hydrogen peroxide. This phenomenon also testified the good specificity of the probe to corresponding ROS. Macrophage RAW 264.7 Cell Extracts Assay. PMA is a well-established ROS stimulator for the cultured macrophages.35 The macrophage RAW 264.7 cell extracts were analyzed under the optimized conditions. On the basis of their migration time, the first and second peaks in Figure 7 were identified as DBO and DCF, respectively. In addition, the two peaks were further identified by standard addition protocol. A typical electropherogram of macrophage RAW 264.7 cell extracts is shown in Figure 7. The concentrations and recoveries of superoxide and hydrogen peroxide in the cell extracts are shown in Table 2. (35) Wrona, M.; Patel, K. B.; Wardman, P. Free Radical Biol. Med. 2005, 38, 262–270.

Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

2197

Table 2. Concentrations and Recoveries of Superoxide and Hydrogen Peroxide in Macrophage RAW 264.7 Cell Extracts (n ) 3) analyte

concn (µM)

added (µM)

found (µM)

mean ± 2SD (µM)

av recovery (%)

ROS (%)

superoxide hydrogen peroxide

0.80 ± 0.06 1.15 ± 0.09

1.0 1.0

1.69, 1.74, 1.80 1.99, 2.06, 2.15

1.74 ± 0.06 2.07 ± 0.08

96.1 93.0

3.4 3.9

CONCLUSIONS A MCE-LIF method was successfully developed for the simultaneous determination of superoxide and hydrogen peroxide in macrophage RAW 264.7 cell extracts. With the use of 0.02 M HEPES running buffer at pH 7.40, the two derivatives, namely, DBO and DCF, were separated in less than 48 s at a voltage of 1800 V. The method is simple, rapid, and sensitive. Most importantly, the method realized the simultaneous separation and determination of superoxide and hydrogen peroxide in a single MCE run. Therefore, it exhibited high application potential in the ROS quantification and the study of biological phenomena and pathophysical mechanisms involving superoxide and hydrogen peroxide.

Natural Science Funds for Distinguished Young Scholar (No. 20725518), the Major Program of National Natural Science Foundation of China (No. 90713019), the National Natural Science Foundation of China (No. 20875058), Science and Technology Development Programs of Shandong Province of China (No. 2008GG30003012), and the Research Foundation for the Doctoral Program of Ministry of Education (No. 20060445002).

ACKNOWLEDGMENT This work was supported by the National Basic Research Program of China (973 Program, 2007CB936000), the National

Received for review August 24, 2008. Accepted January 23, 2009.

2198

Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AC801777C